CN113164519A

CN113164519A - Genetically modified tetra-cistronic system comprising a homing receptor or cytokine and a chimeric antigen receptor for immunotherapy

Info

Publication number: CN113164519A
Application number: CN201980051071.5A
Authority: CN
Inventors: 汉斯·G·克林格曼; 劳伦·H·布瓦塞尔; 约翰·H·李; 南森·T·舒默
Original assignee: NantKwest Inc
Current assignee: ImmunityBio Inc
Priority date: 2018-08-01
Filing date: 2019-08-01
Publication date: 2021-07-23
Also published as: EP3829605A4; JP2023078209A; US11129850B2; US20200101111A1; US20210386785A1; AU2019314455A1; EP3829605A1; US11058723B2; WO2020028656A1; IL280511B2; US20200093863A1; US20200038441A1; KR102653878B1; IL280511B; JP7241161B2; KR20210038637A; IL280511A; CA3106324A1; AU2019314455B2; CA3229942A1

Abstract

Provided herein are modified nucleic acids comprising one or more nucleic acids

A cell, the one or more nucleic acids encoding: i) a homing receptor, ii) an Antigen Binding Protein (ABP) or a Chimeric Antigen Receptor (CAR) that specifically binds to a target antigen, iii) an Fc receptor such as CD16 or CD16-158V, and/or iv) a cytokine, wherein the nucleic acid sequence is operably linked to a promoter. Further provided herein are modified nucleic acids comprising one or more nucleic acids

A cell, the one or more nucleic acids encoding: i) IL-12 and/or TGF- β trap, ii) an Antigen Binding Protein (ABP) or Chimeric Antigen Receptor (CAR) that specifically binds to a target antigen, iii) an Fc receptor such as CD16 or CD16-158V, and/or iv) a cytokine, wherein the nucleic acid sequence is operably linked to a promoter. Also provided are compositions comprising these modifications

Compositions and kits of cells, and methods of using these modified cells for the treatment of cancer.

Description

Genetically modified tetra-cistronic system comprising a homing receptor or cytokine and a chimeric antigen receptor for immunotherapy

This application claims priority from our co-pending U.S. provisional application with serial No. 62/713,264 filed on day 8/1 of 2018, 62/713,278 filed on day 8/1 of 2018, 62/713,310 filed on day 8/1 of 2018, and 62/713,323 filed on day 8/1 of 2018. Each of these applications is incorporated by reference herein in its entirety.

Sequence listing

An ASCII text file of the sequence listing 97KB in size, entitled 104077_0007PCT _ Seq _ listing _ rev004_ ST25, was created at 8 months 1 of 2019, submitted electronically with the present application via EFS-Web, and incorporated by reference in its entirety.

Technical Field

The field of the invention is the engineering of cells that use a cytotoxic activated natural killer cell line (NK-92) as a basis to improve immunotherapy against cancer and tumors.

Background

The background description includes information that may be useful in understanding the present invention. There is no admission that any information provided herein is prior art or relevant to the presently claimed invention, nor that any publication specifically or implicitly referenced is prior art.

All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

Cancer immunotherapy based on adoptively transferred tumor-specific cytotoxic lymphocytes is expected to be useful in treating patients with malignant tumors. Despite this early success in certain cancers, treatment of tumors remains a challenge, primarily due to the immunosuppressive nature of the tumor microenvironment. See Swarts et al "Tumor Microenvironmental Complexity: emerging rolls in Cancer Therapy [ complexity of tumor microenvironment: emerging effects in cancer therapy]"Cancer Res [ Cancer research]Vol 72, pages 2473-2480, 2012. In addition to modified T cells, NK cell-based immunotherapy is being explored. FromNatural Killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Natural Killer (NK) cells, which typically comprise about 10% -15% of circulating lymphocytes, bind to and kill target cells (including virus-infected cells and many malignant cells) that are not specific for an antigen and that have not been immunosensitized as before. Herberman et al, Science]214：24(1981)。

Is a cytolytic cancer cell line that is found in the blood of subjects with non-hodgkin's lymphoma, and then immortalized in vitro.

The cells are derived from NK cells, but lack the major inhibitory receptor displayed by normal NK cells, while retaining most of the activating receptor. However,

the cells do not attack normal cells and do not cause unacceptable immune rejection in humans.

A common driver of lymph node metastasis is hypoxia-driven up-regulation of CCR7, and CCR7 is a chemokine receptor found primarily in naive T cells and dendritic cells. It has previously been demonstrated that upregulation of CCR7 receptors on blood NK cells improves homing of NK cells to the lymph nodes, allowing them to follow the same pathway to reach the lymph node compartment, a common pathway for metastatic spread, but has not been demonstrated in clinically relevant cell lines.

Thus, there remains a need for improved NK cells and NK cell-based therapies, particularly where NK cells home to and modulate the tumor microenvironment.

Disclosure of Invention

Provided herein are modified

Cells comprising nucleic acids encoding various functional elements. The functional element is typically a protein or a polypeptidePeptides that provide specific functions that improve the effectiveness of cells as cell lines for immunotherapy. On the one hand, the method comprises the following steps of,

the cell comprises a nucleic acid construct encoding four functional elements. In some embodiments, the nucleic acid construct comprises a sequence encoding four functional elements operably linked to a promoter (referred to as a "tetra-cistronic construct").

In some embodiments, the first element encoded by the nucleic acid construct is a cytokine that provides for expression of the cytokine, such as IL-2 or IL-15

And (4) selecting cells. Thus, in some embodiments, the nucleic acid encodes a cytokine, such as IL-2 or IL-15. In one embodiment, IL-2 is expressed with a signal sequence that directs IL-2 to the endoplasmic reticulum IL-2 ("ERIL-2"). In another embodiment, IL-15 is expressed with a signal sequence that directs IL-15 to the endoplasmic reticulum IL-15 ("ERIL-15").

In some embodiments, the second element encoded by the nucleic acid construct is an Fc receptor. In some embodiments, the Fc receptor is an Fc-gamma receptor (Fc γ R). In some embodiments, the Fc-gamma receptor is Fc γ RIII-a (also known as CD16), which is a low affinity Fc receptor that binds to IgG antibodies and activates ADCC. In some embodiments, the CD16 receptor comprises a phenylalanine (F) -valine (V) substitution at amino acid position 158(F158V) of the mature form of the polypeptide (SEQ ID NO: 12), which corresponds to position 176 of the full-length form of the polypeptide comprising the signal sequence. In one embodiment, the Fc receptor comprises SEQ ID NO: 13 or the nucleic acid sequence of SEQ ID NO: 12.

In some embodiments, the first element and the second element are present in the nucleic acid construct. Thus, in some embodiments, the nucleic acid construct encodes an Fc receptor (such as CD16) and erll-2.

In some embodiments, the third element encoded by the nucleic acid construct is a homing receptor. In some embodimentsThe homing receptor is a cytokine receptor, a G protein-coupled receptor, a chemokine receptor, a cell adhesion molecule, a selectin, or an integrin. In some embodiments, the homing receptor is operably linked to a promoter that allows transcription of the nucleic acid. Modified

Cells are able to migrate towards a source of chemokines, which are ligands for receptors. Modified unlike normal blood-derived NK cells

Cells can be developed into cell lines relevant to human clinical trials, providing significant advantages for immunotherapy. Examples of homing receptors include G protein-coupled receptors, such as chemokine receptors, including but not limited to CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC or CXCL14 receptors; a cytokine receptor; cell adhesion molecules such as selectins, including L-selectin (CD 62L); integrins such as α 4 β 7 integrin, LPAM-1 and LFA-1. In some embodiments, the homing receptor is a cell adhesion molecule, such as LFA-1. In some embodiments, the homing receptor is a selectin, such as L-selectin (CD 62L). In some embodiments, the homing receptor is an integrin, such as α 4 β 7 integrin, LPAM-1 or VLA-4. In some embodiments, the homing receptor is a C-C or C-X-C chemokine receptor. Thus, in some embodiments, the third element encoded by the nucleic acid is a homing receptor as described herein.

In some embodiments, the third element encoded by the nucleic acid construct is a secreted cytokine, whereby the cytokine enhances or improves

The function of cells as immunotherapeutic agents. The cytokine may also modulate the tumor microenvironment. In some embodiments, the secreted cytokine that modulates the tumor microenvironment is IL-12 or IFN- α. Thus, in someIn embodiments, the third element encoded by the nucleic acid construct is a cytokine, such as IL-12 or IFN- α.

Thus, in some embodiments, the third element encoded by the nucleic acid construct is a chemokine, such as XCL1, CCL5, CCL21, or CCL 16. In some embodiments, the third element encoded by the nucleic acid construct is a Toll-like receptor (TLR) agonist.

In one aspect, the third element encoded by the nucleic acid construct is IL-12.

TGF- β expression within tumors is known to inhibit the anti-tumor activity of leukocytes in the tumor microenvironment. Thus, in some embodiments, the third element encoded by the nucleic acid construct is a TGF- β inhibitor, e.g., a peptide that inhibits TGF- β. In some embodiments, the third element encoded by the nucleic acid construct is a TGF- β trap. In some embodiments, the TGF- β trap comprises the extracellular domain of a TGF β RII molecule. In some embodiments, the TGF- β trap comprises a single-chain dimer of the extracellular domain of a TGF β RII molecule, and most preferably comprises a single-chain dimer of the extracellular domain of TGF- β receptor II.

In some embodiments, the NK cells described herein are administered with a TGF- β inhibitor to block TGF- β and help remove immunosuppression. In some embodiments, the NK cells described herein are administered with other immunotherapy to help reduce or eliminate tumors. For example, TGF-. beta.can be inhibited by intratumoral injection of an inhibitory peptide in combination with intratumoral injection of poly (I: C) and alpha-CD 40 antibodies. In some embodiments, a TGF- β inhibitor is combined with IL-2.

In some embodiments, the fourth element encoded by the nucleic acid construct is an antigen binding protein ("ABP"). In some embodiments, the antigen binding protein specifically binds to a tumor associated antigen. In some embodiments, the ABP comprises a fragment of an antibody, such as an scFv. In some embodiments, the antigen binding protein comprises or is part of a Chimeric Antigen Receptor (CAR). In some embodiments, the nucleic acid encodes an ABP or CAR that specifically binds: CD19, CD20, NKG2D ligand, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, CSPG-4, IGF1-R, Flt-3, CD276, CD123, PD-L1, BCMA, CD33, B7-H4 or 41 BB.

In another aspect, modified

The cell comprises a nucleic acid encoding IL-12. In another aspect, modified

The cells contain nucleic acid encoding a TGF- β trap. In some embodiments, the TGF- β trap comprises a single-chain dimer of the extracellular domain of a TGF β RII molecule.

In another aspect, modified

The cell comprises a nucleic acid encoding a cytokine that provides for expression of the cytokine

Selection of cells or allowing expression of the cytokine

The cells survive. In some embodiments, the nucleic acid encodes a cytokine, such as IL-2 or IL-15. In one embodiment, IL-2 is expressed with a signal sequence that directs IL-2 to the endoplasmic reticulum IL-2 ("ERIL-2"). In one embodiment, IL-15 is expressed with a signal sequence that directs IL-15 to the endoplasmic reticulum IL-2 ("ERIL-15").

In some embodiments, modified

The cell comprises a nucleic acid encoding an Fc receptor. In some embodiments, the Fc receptor is an Fc-gamma receptor (Fc γ R). In some embodiments, the Fc-gamma receptor is Fc γ RIII-a (also known as CD16), which is a low affinity Fc receptor that binds to IgG antibodies and activates ADCC. In some embodimentsThe CD16 receptor contained a phenylalanine (F) -valine (V) substitution at amino acid position 158(F158V) of the mature form of the polypeptide (SEQ ID NO: 12), which corresponds to position 176 of the full-length form of the polypeptide containing the signal sequence. In one embodiment, the Fc receptor comprises SEQ ID NO: 13 or the nucleic acid sequence of SEQ IID NO: 12.

In some embodiments, modified

The cells comprise a nucleic acid encoding an antigen binding protein ("ABP"). In some embodiments, the antigen binding protein specifically binds to a tumor associated antigen. In some embodiments, the ABP comprises a fragment of an antibody, such as an scFv. In some embodiments, the antigen binding protein comprises or is part of a Chimeric Antigen Receptor (CAR). In some embodiments, the nucleic acid encodes an ABP or CAR that specifically binds: CD19, CD20, NKG2D ligand, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, CSPG-4, IGF1-R, Flt-3, CD276, CD123, PD-L1, BCMA, CD33, B7-H4 or 41 BB.

In another aspect, modified

The cells comprise nucleic acids encoding secreted cytokines that regulate the tumor microenvironment. In some embodiments, the cytokine that modulates the tumor microenvironment is a chemokine, such as XCL1, CCL5, CCL21, or CCL 16. In some embodiments, modified

The cells comprise a nucleic acid encoding a Toll-like receptor (TLR) agonist. In some embodiments, modified

The cells comprise a nucleic acid encoding IL-12 or IFN- α. In some embodiments, modified

The cells contain nucleic acids encoding TGF-beta inhibitors (e.g., peptides that inhibit TGF-beta). In some embodiments, modified

The cells contain nucleic acid encoding a TGF- β trap. In some embodiments, the TGF- β trap comprises the extracellular domain of a TGF β RII molecule, or a single chain dimer of the extracellular domain of a TGF β RII molecule.

In one aspect, modified

The cells comprise one or more, or more nucleic acid molecules encoding a homing receptor, ABP or CAR, Fc receptor and/or cytokine that provides for expression of the cytokine

Selection of cells or allowing expression of the cytokine

The cells survive. Thus, in some embodiments, modified

The cell comprises a nucleic acid molecule encoding a chemokine receptor, CAR, CD16, and erll-2. In some embodiments, modified

The cells comprise a nucleic acid molecule encoding CCR7 or CXCR2, CAR, CD16, and erll-2. In some embodiments, modified

The cells comprise a nucleic acid molecule encoding IL-12 or TGF- β trap, CAR, CD16, and erll-2.

In some embodiments, the CAR comprises a cell from fce receptor gamma (fce RI gamma)An intracellular signaling domain. In one embodiment, the CAR is comprised of

The cells are transiently expressed. In one embodiment, the CAR is comprised of

The cells are stably expressed.

Heretofore, Fc ε RI γ -containing CAR has not been

Cells, other NK cell lines, or endogenous NK cells, since other signaling domains (e.g., CD3 ζ) were identified to be more potent, particularly when combined with additional signaling domains (second generation CARs and third generation CARs). Described herein is the surprising discovery that expression of "first generation" CARs comprising an intracellular domain from fcsri γ

Of cells with CARs expressing a CD3 zeta signaling domain alone or in combination with other signaling domains (i.e., second generation or third generation CARs)

The cell has the same or higher cytotoxic activity against a cancer cell expressing an antigen recognized by the CAR as compared to the cell. In one embodiment, a CD3 zeta signaling domain contemplated herein may comprise a sequence identical to SEQ ID NO: 40, or a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

In one aspect, a method is described

Cells or cell lines of

Surface of cellA Chimeric Antigen Receptor (CAR), wherein the CAR comprises a cytoplasmic domain of fcsri γ. In one embodiment, the cytoplasmic domain of fcsri γ comprises a sequence identical to SEQ ID NO: 31 with at least 95% sequence identity.

In some embodiments, the cytoplasmic domain of fcsri γ is encoded by a sequence identical to SEQ ID NO: 32 has at least 95% sequence identity.

In some embodiments, the CAR comprises a hinge region from CD 8. In some embodiments, the CAR comprises a transmembrane domain from CD 28.

In some embodiments, the nucleic acid sequence is encoded by a nucleic acid comprising SEQ ID NO: 31 (fcsri γ intracellular cytoplasmic domain), SEQ ID NO: 32 (fceri γ intracellular signaling domain minus transmembrane domain), SEQ ID NO: 33(CD8 hinge region), SEQ ID NO: 34(CD8 hinge region DNA), SEQ ID NO: 35(CD28 transmembrane domain) and/or SEQ ID NO: 36(CD28 transmembrane domain, minus ITAM or intracellular sequence)

The cells or cell lines are genetically modified. In one embodiment, the amino acid sequence of the CD8 hinge region, CD28 transmembrane and FceRI γ signaling domain comprises an amino acid sequence identical to SEQ ID NO: 37 or SEQ ID NO: 38, or a polypeptide or polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In some embodiments, the nucleic acid construct further comprises a promoter that facilitates transcription of the nucleic acid sequence. In some embodiments, the promoter is an inducible promoter. In some embodiments, the nucleic acid construct is a polycistronic vector comprising one or more Internal Ribosome Entry Sites (IRES) to effect translation initiation from an internal region of an mRNA transcribed from the nucleic acid sequence. In some embodiments, the nucleic acid construct comprises a sequence encoding a 2A peptide, such as a T2A, P2A, E2A, or F2A peptide, to produce equimolar levels of the polypeptide encoded by the same mRNA. In some embodiments, the nucleic acid construct further comprises a nucleic acid sequence encoding an Antigen Binding Protein (ABP). In some embodiments, the ABP is a scFv or a codon optimized scFv. In some embodiments, the ABP is specificSexually binds to an antigen expressed by the tumor cell. In some embodiments, the ABP is part of a Chimeric Antigen Receptor (CAR). In some embodiments, the construct comprises a nucleic acid encoding a cytokine that provides for expression of the cytokine (such as IL-2)

Selection of cells or allowing thereof

The cells survive. In one embodiment, the cytokine is targeted to the endoplasmic reticulum. In one embodiment, the CAR scFv can comprise an amino acid sequence identical to SEQ ID NO: 39, or a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

In some embodiments, the construct comprises the vector shown in fig. 10. In some embodiments, for

The cells or cell lines are genetically modified to express CD16 on the cell surface. In one embodiment, the pair

The cells or cell lines are genetically modified to express high affinity CD16 on the cell surface (F158V).

In one embodiment, the ABP or CAR targets or specifically binds a tumor associated antigen. In one embodiment, the tumor associated antigen is selected from the group consisting of: CD19, CD20, NKG2D ligand, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, CSPG-4, IGF1-R, Flt-3, CD276, CD123, PD-L1, BCMA, CD33B7-H4 and 41 BB. In one embodiment, the tumor associated antigen is CD 19. In another embodiment, the tumor associated antigen is CD 33.

In one aspect, the disclosure relates to a method of making a lens assembly

A cell line transformed with a nucleic acid encoding a Chimeric Antigen Receptor (CAR) having a cytoplasmic domain of fcsri γ, wherein the CAR is in

Expressed on the surface of the cell. In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is DNA.

In some embodiments of the present invention, the,

the cells are further modified to express at least one cytokine or variant thereof that provides for expression of the cytokine

Selection of cells or allowing expression of the cytokine

The cells survive. In one embodiment, the at least one cytokine is derived from

The cells are transiently expressed. In one embodiment, the at least one cytokine is derived from

The cells are stably expressed.

In some embodiments, modified

The cell comprises an expression vector comprising one or more of the nucleic acid molecules described herein. In some embodiments, the nucleic acid molecule is operably linked to a promoter capable of initiating transcription of the nucleic acid molecule. In some embodiments, each nucleic acid molecule of the plurality of nucleic acid molecules is associated with a separate, distinct, and/or different promoterAre operatively connected. In some embodiments, one or more of these nucleic acid molecules are operably linked to the same promoter. In one embodiment, the nucleic acid molecules encoding the homing receptor, CAR, Fc receptor, and cytokine are operably linked to the same promoter or a single promoter. In some embodiments, the promoter is an inducible promoter. In one embodiment, the cytokine-encoding nucleic acid molecule is located downstream or 3' of the nucleic acid molecule encoding the homing receptor, CAR, and Fc receptor (e.g., CD16 or high affinity CD 16).

In some embodiments of the present invention, the,

the cell expresses a protein encoded by a nucleic acid molecule described herein on the surface of the cell. For example, in some embodiments, modified

Cells express homing receptors, ABPs or CARs and Fc receptors (e.g., CD16 or high affinity CD16) on the cell surface.

Also provided are compositions comprising the modified

Compositions and kits for cells. Methods of making the modified cells and methods of using these cells to treat cancer are provided.

In another aspect, methods for treating cancer or reducing tumor size are described. In some embodiments, a method of treating cancer or reducing tumor size comprises administering to a subject in need thereof a therapeutically effective amount of a modified antibody described herein

A cell, wherein the administration treats cancer or reduces tumor size in the subject. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of the modified

Cells, modified

The cell comprises a nucleic acid encoding a homing receptor, an ABP or CAR that specifically binds to a target antigen, an Fc receptor (such as CD16 or CD16-158V), and/or a cytokine (such as erll-2 or erll-15). In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of the modified

Cells, modified

The cells comprise a nucleic acid encoding a secreted cytokine, an ABP or CAR that specifically binds to a target antigen, an Fc receptor (such as CD16 or CD16-158V), and/or a cytokine (such as erll-2 or erll-15).

In another aspect, there is provided the use of a composition described herein for the treatment of a disease. In some embodiments, modified as described herein are provided

The cells are useful as a medicament for treating diseases. In some embodiments, modified as described herein are provided

The cells are useful for treating diseases. In some embodiments, modified

The cell comprises a nucleic acid encoding a homing receptor, an ABP or CAR that specifically binds to a target antigen, an Fc receptor (such as CD16 or CD16-158V), and/or a cytokine (such as erll-2 or erll-15). In some embodiments, modified

The cells comprise a nucleic acid encoding a secreted cytokine, an ABP or CAR that specifically binds to a target antigen, an Fc receptor (such as CD16 or CD16-158V), and/or a cytokine (such as erll-2 or erll-15). In some embodiments, the disease is cancer.

The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

Drawings

FIG. 1 is a view showing a view for insertion

Schematic representation of plasmid pNKAT-CCR7-LP3 containing the CCR7 receptor at the AAVS1 locus in cells.

FIG. 2 is a schematic diagram showing plasmid pCRENFAT-CCL21 containing NFAT-responsive CCL21 gene.

FIG. 3 is a schematic view of a display

Cell-associated phenotypic markers in wild type

Cells and modified expressing CCR7

Graph of expression in cells. (Lane 1: aNK (wild type); Lane 2: modified

Cells (MA 3); lane 3: modified

Cells (MB 4); lane 4: modified

Cells (MB 6); lane 5: modified

Cells (ME 6); lane 6: modified

Cells (MH 3); a: isotype (APC); b: CD54 (ICAM-1); c: NKp 30; d: NKG2D

FIG. 4 is a schematic showing the modification of expression of CCR7

Graph of the cytotoxic activity of cells against K562 cells.

FIG. 5 is a schematic showing the modification of expression of CCR7

Graph of the cytotoxic activity of cells against HL-60 cells.

FIGS. 6A and 6B are graphs showing NFAT-luciferase reporter activation in NK-92 cells confirmed in the context of binding to K562 and SUP-B15 cells (when NK-92 cells were electroporated with mRNA of CD 19-CAR).

FIG. 7 is a schematic showing the modification of expression of CCR7

Graph of migration of cells (Mi-aNK) to chemokines CCL19 and CCL 21.

FIG. 8 shows a schematic representation of modified antibodies described herein for in vitro testing

Schematic representation of an exemplary method of a cell. Activated

Cells (anks) are modified to express chemokine receptors (e.g., CCR7) and target cells are modified to express a chemokine that binds to the receptor (e.g., CCL19 or CCL 21). As shown, the modified in a modified Boyden Chamber Transwell assay

The cells were tested.

FIG. 9 shows the use of the modified as described herein

Representative cytotoxicity assays of cells. The modified from FIG. 8 was tested

Cytotoxicity of cells against K562 target cells expressing and secreting one or two chemokine ligands. The ML4 clone showed the highest percent target cell lysis and increased when K562 target cells expressed both CCL19 and CCL 21.

FIG. 10 is a schematic showing plasmid pNKAT-CCR7-CD19CAR-CD16-ERIL2 (referred to as a "tetra-cistronic vector") that can be used to stably transfect cells at a single insertion site.

FIG. 11 is a schematic showing the linearized plasmid from FIG. 10.

FIG. 12 shows

Cell surface expression of CCR7, CD16, and CD19 CARs by cells. "aNK" is wild type

A cell line. "ML 4" is an aNK cell line (i.e., Mi-aNK) transfected with a nucleic acid construct encoding CCR7 operably linked to a promoter. "P2" is an aNK cell line transfected with nucleic acid constructs encoding CCR7, CD16, ER-IL2, and CD19CAR (i.e., Mi-T-haNK).

Figure 13 homing of non-CR and Mi-T-haNK cells to the parental or CCL 19-expressing tumor at the indicated hours after NK cell administration. Data are mean ± SEM. The-and + -signs indicate the expression status of the CCR7 receptor (first symbol) and the CCL19 ligand (second symbol). P < 0.05 was determined by one-way ANOVA followed by multiple comparisons by Tukey test. The last diagram presents a time course curve.

Figure 14. non-CR and Mi-T-haNK cells from a single animal were compared head-to-head to infiltration of parental or CCL 19-expressing tumors 24 hours after dosing. 3 of the 4 animals that received Mi-T-haNK cells showed a higher infiltration into CCL19+ tumors, while 3 of the 4 animals that received non-CR CD19T-haNK cells showed similar levels of infiltration to K562 and K-19 tumors. One "outlier" animal in each group is indicated by a dashed line.

FIG. 15 illustrates the survival curves of IV Raji-19.5 tumor-bearing animals. Survival curves of Raji-19.5 IV tumor-bearing NSG mice treated with vehicle, CD19t-hanK cells or R7-19.1 cells. Statistical analysis was performed by the log-rank (Mantel-Cox) test. 0.0002 ═ P; p < 0.0001.

FIG. 16 illustrates weight changes in the IV Raji-19.5 tumor model. Weight change curves (% change in body weight compared to day 0) for IV Raji-19.5 tumor-bearing animals treated with vehicle, CD19t-hanK cells or R7-19.1 cells. Data are mean ± SEM. Red arrows indicate days of administration. Weight measurements made on the day of dosing were made prior to dose administration. For all time points before day 20, the curves for the NK cell treated group reached statistically significant differences (P < 0.05) compared to the vehicle control group by two-way analysis of variance (2-way ANOVA) followed by multiple comparisons by Tukey's test.

FIG. 17 illustrates the size of SC Raji-19.5 tumors at random grouping. Individual tumor sizes at random grouping are shown. The size of the black square frame is more than 200mm³And blue square surrounding < 200mm³Small tumors of (2). Group mean ± SEM are also shown.

FIG. 18 illustrates tumor growth of a large tumor subpopulation of SC Raji-19.5 tumor-bearing mice. (A) And (4) group analysis. Data are mean ± SEM. Statistical analysis was performed using a two-way mixed-effects analysis (2-way mixed-effects analysis) followed by multiple comparisons by Tukey's test. Statistical significance was not achieved. (B) Individual curves. Red arrows indicate days of administration. Tx: and (6) processing.

FIG. 19 illustrates tumor growth of a small tumor subpopulation of SC Raji-19.5 tumor-bearing mice. (A) And (4) group analysis. Data are mean ± SEM. Statistical analysis was performed using a two-way mixed effects analysis followed by multiple comparisons by Tukey test. No statistical significance was detected between any 2 groups at any time point. (B) Individual curves. Red arrows indicate days of administration. Tx: and (6) processing.

FIG. 20 illustrates weight changes in the SC Raji-19.5 tumor model. Weight change curves (% change in body weight compared to day 1) for SC Raji-19.5 tumor-bearing animals treated with vehicle, CD19t-hanK cells or R7-19.1 cells. Data are mean ± SEM. Red arrows indicate days of administration. Body weight measurements made on the day of dosing were made prior to dose administration. For all time points before day 16, the curves for the NK cell treated group reached statistically significant differences compared to the vehicle control group by two-way mixed effect analysis followed by multiple comparisons by Tukey test.

Figure 21 illustrates one embodiment of a PD-L1 CAR construct equipped with a tetra-cistronic TGF β -trap.

FIG. 22 illustrates expression analysis of PD-L1 CAR and CD16 in PD-L1(TGF β -trap) t-haNK clones.

FIG. 23 illustrates secretion of TGFb trap into the culture supernatant of TGF β trap/PD-L1 t-haNK clone.

FIG. 24 illustrates the cytotoxicity of the tetra-cistronic TGF-beta-trap construct on K562 target cells.

Figure 25 illustrates CAR killing of SUP-B15 target cells expressing PD-L1 by the tetra-cistronic TGF β -trap construct.

FIG. 26 illustrates CAR killing of MDA-MB 231 target cells by the tetra-cistronic TGF-beta-trap construct.

FIG. 27 illustrates the tetra-cistronic TGF-beta-trap construct pair SUP-B15^CD19-CD20+ADCC of (1).

FIG. 28 illustrates TGF β/SMAD luciferase reporter HEK293 cells induced by TGF β.

Figure 29 illustrates secreted TGF β -trap chelated TGF β and inhibited luciferase expression in the HEK293T reporter assay.

FIG. 30 illustrates IL-12 viral transduction

IL-12 secretion from cell lines.

FIG. 31 illustrates an embodiment of a tetra-cistronic IL-12/PD-L1 t-hanK construct.

FIG. 32 illustrates cytotoxicity data for CCR7 CD19t-hanK cells.

FIG. 33 illustrates IL-12/PD-L1 t-haNK^TMIL-12 secretion from cell lines.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of immunology and immunotherapy.

In this specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

All numerical designations (e.g., pH, temperature, time, concentration, amount, and molecular weight, including ranges) include changes commonly encountered by one of ordinary skill in the art. Thus, depending on the associated significand, the value may include a change in (+) or (-) increment of 0.1 or 1.0, as appropriate. It is to be understood that, although not always explicitly stated, all numerical designations may be preceded by the term "about". As used herein, the term "about" may also mean that the value may vary by ± 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

It is also to be understood that, although not always explicitly stated, the reagents described herein are exemplary only and equivalents thereof are known in the art.

"optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

The term "comprising" is intended to mean that the compositions and methods include the recited elements, but not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean to exclude other elements of any significance to the combination. For example, a composition consisting essentially of the elements defined herein shall not exclude other elements that do not materially affect the basic and novel characteristics of the claimed invention. "consisting of" shall mean excluding more than trace amounts of other ingredients and the substantial method steps recited. Embodiments defined by each of these transition terms are within the scope of this disclosure.

The term "homing receptor" refers to a receptor that activates a cellular pathway that directly or indirectly results in the migration of a cell to a target cell or tissue. For example, homing receptors expressed by leukocytes are used by leukocytes and lymphocytes to enter secondary lymphoid tissue via the high endothelial venules. Homing receptors can also be used by cells to migrate toward chemical gradient sources, such as chemokine gradient sources. Examples of homing receptors include G protein-coupled receptors, such as chemokine receptors, including but not limited to CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, and DARC; a cytokine receptor; cell adhesion molecules such as selectins, including L-selectin (CD 62L); integrins such as α 4 β 7 integrin, LPAM-1 and LFA-1. Homing receptors typically bind to cognate ligands on target tissues or cells. In some embodiments, the homing receptor binds to Addressin (Addressin) on the endothelium of the venules, such as the mucosal angiotensin protein cell adhesion molecule 1 (MAdCAM-1).

As used herein, "immunotherapy" refers to modified or unmodified, alone or in combination, and capable of inducing cytotoxicity upon contact with a target cell

Use of cells, naturally occurring or modified NK cells or T cells.

As used herein, a "Natural Killer (NK) cell" is a cell of the immune system that kills a target cell in the absence of specific antigen stimulation, and is not limited according to the Major Histocompatibility Complex (MHC) class. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers.

The term "endogenous NK cell" is used to refer to a donor (or patient) -derived NK cell, with

The cell lines are different. Endogenous NK cells are typically heterogeneous populations of cells in which NK cells have been enriched. Endogenous NK cells may be used for autologous or allogeneic treatment of a patient.

The term "NK-92" refers to a highly potent and unique cell line (the right of which is assigned to) derived from Gong et al (1994)

All) natural killer cells (hereinafter referred to simply as "

Cells "). Immortalized NK cell lines were originally obtained from patients with non-hodgkin's lymphoma. Unless otherwise indicated, terms

Is referred to as original

Cell lines and modified (e.g. by introduction of foreign genes)

A cell line.

Cells and exemplary and non-limiting modifications thereof are described in U.S. Pat. Nos. 7,618,817, 8,034,332, 8,313,943, 9,181,322, 9,150,636 and published U.S. application No. 10/008,955, which are all incorporated herein by reference in their entirety, including wild-type

(F176V)、

And

cells are known to those of ordinary skill in the art, and to those of ordinary skill in the art, such cells are available from southwest corporation (NantKwest, Inc).

The term "aNK" refers to unmodified natural killer cells (hereinafter abbreviated) derived from a highly potent unique cell line described by Gong et al (1994), which is owned by southernwestern Western company "

Cells "). The term "haNK" refers to natural killer cells (hereinafter abbreviated) derived from a highly potent unique cell line described by Gong et al (1994), which is owned by southernwestern western company, modified to express CD16 on the cell surface "

Cell "or"

Cells "). In some embodiments, CD16+

Cells contain a high affinity CD16 receptor on the cell surface. The term "taNK" refers to a natural killer cell derived from a highly potent unique cell line described by Gong et al (1994), which is owned by southernwestern western company, modified to express a chimeric antigen receptor (hereinafter referred to as "CAR-modified

Cell "or"

Cells "). The term "t-haNK" refers to natural killer cells derived from a highly potent and unique cell line described by Gong et al (1994) (which is owned by southwestern corporation) modified to express CD16 on the cell surface and to express a chimeric antigen receptor (hereinafter referred to as "CAR-modified CD16 +)

Cells "or" t-haNK cells "). In some embodiments, t-haNK cells express the high affinity CD16 receptor on the cell surface.

The terms "chemokine-targeted T-haNK" and "Mi-T-haNK" refer to T-haNK cells modified to express chemokine receptors on the cell surface.

As used herein, the terms "cytotoxic" and "cytolytic" when used to describe effector cells (such as

Cell) is intended to be synonymous. In general, cytotoxic activity involves killing of target cells by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more specifically refers to the activity of an effector to lyse the plasma membrane of a target cell, thereby disrupting its physical integrity. This results in killing of the target cells. Without wishing to be bound by theory, it is believed that

The cytotoxic effect of the cells isDue to cell lysis.

The term "killing" with respect to a cell/cell population is intended to include any type of manipulation that will result in the death of that cell/cell population.

The term "Fc receptor" refers to a protein found on the surface of certain cells (e.g., natural killer cells) that contributes to the protective function of immune cells by binding to the portion of an antibody referred to as the Fc region. Binding of the Fc region of an antibody to the Fc receptor (FcR) of a cell stimulates phagocytic or cytotoxic activity of the cell through antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcR is classified according to the type of antibody it recognizes. For example, Fc-gamma receptors (Fc γ R) bind IgG class antibodies. FC γ RIII-a (also known as CD16) is a low affinity FC receptor that binds to IgG antibodies and activates ADCC. FC γ RIII-A is commonly found on NK cells.

Cells did not express FC γ RIII-A. The Fc-epsilon receptor (Fcepsilon R) binds to the Fc region of IgE antibodies.

As used herein, the term "chimeric antigen receptor" (CAR) refers to an extracellular antigen-binding domain fused to an intracellular signaling domain. The CAR can be expressed in T cells or NK cells to increase cytotoxicity. Typically, the extracellular antigen-binding domain is an scFv specific for an antigen found on a target cell. Based on the specificity of the scFv Domain, expression of CAR

Cells target cells that express certain antigens on the cell surface. The scFv domains can be engineered to recognize any antigen, including tumor-specific antigens. For example, CD19 CARs recognize CD19, and CD19 is a cell surface marker expressed by certain cancers.

The term "tumor-specific antigen" as used herein refers to an antigen that is present on a cancer cell or neoplastic cell but is not detectable on normal cells derived from the same tissue or lineage as the cancer cell. As used herein, a tumor-specific antigen also refers to a tumor-associated antigen, i.e., an antigen that is expressed at a higher level on cancer cells as compared to normal cells derived from the same tissue or lineage as the cancer cells.

The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or analogs thereof. The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), an exon, an intron, messenger RNA (mrna), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. Nucleotide structural modifications, if present, may be made before or after polynucleotide assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of a polynucleotide of the invention encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to make up the double-stranded form.

A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (a); cytosine (C); guanine (G); thymine (T); when the polynucleotide is RNA, uracil (U) replaces thymine. Thus, the term "polynucleotide sequence" is a letter representation of a polynucleotide molecule.

"homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Sequence similarity can be determined by comparing the position in each sequence, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The percentage of sequence similarity between sequences is a function of the number of matching or homologous positions that the sequences share within a given comparison window. The sequence may be at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence described herein.

The term identical or percent identity, in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same, or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity over a specified region when compared and aligned for maximum correspondence over a comparison window or specified region), as measured using the default parameters described below using the BLAST or BLAST 2.0 sequence comparison algorithm, or as measured by manual alignment and visual inspection (see, e.g., NCBI website, etc.). Such sequences are then said to be substantially identical. This definition also refers to or can be applied to the complementary sequence of the test sequence. The definition also includes sequences with deletions and/or additions, as well as those with substitutions. As described below, the preferred algorithm may take into account vacancies, etc. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. When using the sequence comparison algorithm, inputting the test sequence and the reference sequence into a computer; if necessary, designating subsequence coordinates; and specifying the sequence algorithm program parameters. Preferably, default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

As used herein, a comparison window includes reference to a segment having a number of any one consecutive position selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, after optimal alignment of two sequences, one sequence can be compared to a reference sequence having the same number of consecutive positions. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by: by the local homology algorithm of Smith and Waterman, adv.appl.math. [ apply mathematical progression ] 2: 482 (1981); biol. [ journal of molecular biology ] 48: 443 (1970); similarity search method by Pearson and Lipman, proc.nat' l.acad.sci.usa [ journal of american national academy of sciences ] 85: 2444 (1988); performing these algorithms by computerization (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 scientific Phd., Wisconsin Madison, Wis.); or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology, latest Protocols in Molecular Biology (edited by Ausubel et al, journal of 1995)).

Preferred examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, respectively, described in Altschul et al, nuc.acids Res. [ nucleic acid research ] 25: 3389-: 403-. Percent sequence identity for a nucleic acid or protein is determined using BLAST and BLAST 2.0 using the parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information, as is known in the art. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of a selected length (W) in the query sequence that match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hit points serve as seeds for initiating searches to find longer HSPs containing them. Word hit points extend in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, cumulative scores can be calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of word hit points to each direction terminates when the following occurs: the cumulative alignment score decreased by an amount X from its maximum realizations; the cumulative score becomes zero or lower due to accumulation of one or more negative scoring residue alignments; or to the end of either sequence. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The expected value (E) represents the number of different alignments that score equal to or higher than the expected score that would occur by chance in a database search. The BLASTN program (for nucleotide sequences) defaults to use a word length (W) of 11, an expectation (E) of 10, M-5, N-4, and a comparison of the two strands. For amino acid sequences, the BLASTP program defaults to a word length of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, proc. natl. acad. sci. usa [ proceedings of the american academy of science ] 89: 10915, 1989) alignment (B) of 50, an expectation (E) of 10, M-5, N-4.

The term transformation as used herein refers to the process of introducing an exogenous or heterologous nucleic acid molecule (e.g., a vector or recombinant nucleic acid molecule) into a recipient cell. The exogenous or heterologous nucleic acid molecule may or may not be integrated (i.e., covalently linked) into the chromosomal DNA that makes up the genome of the host cell. For example, the exogenous or heterologous polynucleotide may be maintained on an additional element such as a plasmid. Alternatively or additionally, the exogenous or heterologous polynucleotide may be integrated into the chromosome so that it is inherited by daughter cells by chromosomal replication. Methods of transformation include, but are not limited to, calcium phosphate precipitation; fusing the recipient cell with a bacterial protoplast comprising the recombinant nucleic acid; treating the recipient cell with a liposome comprising the recombinant nucleic acid; DEAE dextran; fusion using polyethylene glycol (PEG); electroporation; magnetic perforation (magnetophoresis); biolistic delivery; a retroviral infection; lipofection; and microinjection of DNA directly into cells.

As used with respect to cells, the term "transformed" refers to cells that have undergone transformation as described herein, such that the cells carry exogenous or heterologous genetic material (e.g., recombinant nucleic acids). The term "transformed" may also or alternatively be used to refer to cells, cell types, tissues, organisms, etc., that contain exogenous or heterologous genetic material.

As used herein with respect to introducing nucleic acids into a cell or organism, the term "introducing" is intended to have the broadest meaning and is intended to encompass introduction by, for example, transformation methods (e.g., calcium chloride-mediated transformation, electroporation, particle bombardment) and also encompasses introduction by other methods including transduction, conjugation, and mating. Optionally, the nucleic acid is introduced into the cell or organism using a construct.

The terms "modified" and "recombinant" when used in reference to a cell, nucleic acid, polypeptide, vector, etc., indicate that the cell, nucleic acid, polypeptide, vector, etc., has been modified or is the result of a laboratory procedure and is not naturally occurring. Thus, for example, a modified cell includes a cell produced or modified by a laboratory method, e.g., by a transformation method used to introduce a nucleic acid into a cell. The modified cell may comprise a nucleic acid sequence not found in the native (non-recombinant) form of the cell, or may comprise a nucleic acid sequence that has been altered, for example, linked to a non-native promoter.

As used herein, the term "exogenous" refers to a substance, such as a nucleic acid (e.g., a nucleic acid including regulatory sequences and/or genes) or polypeptide, that is artificially introduced into a cell or organism and/or does not naturally occur in the cell in which it is located. In other words, a substance such as a nucleic acid or polypeptide originates from outside the cell or organism into which it is introduced. The exogenous nucleic acid can have the same nucleotide sequence as a nucleic acid that naturally occurs in a cell. For example, can be

Cell engineering to include

Nucleic acids of sequence, for example heparanase. Optionally, endogenous to

The heparanase sequence is operably linked to a gene having regulatory sequences not involved in natural conditions. Although it is not limited to

The heparanase sequence may be naturally present in the host cell, but according to the present disclosure, the introduced nucleic acid is exogenous. The exogenous nucleic acid may have a nucleotide sequence that is different from any nucleic acid naturally present in the cell. For example, the exogenous nucleic acid may be a heterologous nucleic acid, i.e., a nucleic acid from a different species or organism. Thus, the exogenous nucleic acid may have a nucleic acid sequence that is identical to the nucleic acid naturally found in the source organism, but that is different from the cell into which it is introduced. As used herein, the term "endogenous" refers to a nucleic acid sequence that is native to a cell. As used herein, the term "heterologous" refers to a nucleic acid sequence that is not native to the cell, i.e., is from an organism that is different from the cell. The terms "exogenous" and "endogenous" or "heterologous" are not mutually exclusive. Thus, a nucleic acid sequence may be both exogenous and endogenous, meaning that the nucleic acid sequence may be introduced into a cell, but has a sequence that is the same as or similar to a nucleic acid sequence naturally occurring in the cell. Similarly, a nucleic acid sequence may be exogenous and heterologous, meaning that the nucleic acid sequence may be introduced into a cell, but has a sequence that is not native to the cell, e.g., a sequence from a different organism.

As used herein, a control or standard control refers to a sample, measurement, or value that is used as a reference (typically a known reference) for comparison to a test sample, measurement, or value. For example, a test cell, e.g., a cell transformed with a nucleic acid sequence encoding an Fc receptor gene, can be compared to a known normal (wild-type) cell (e.g., a standard control cell). The standard control may also represent an average measurement or mean value collected from a population of cells that do not express Fc receptors or have Fc receptor activity or have minimal levels of Fc receptor activity (e.g., standard control cells). The skilled artisan will recognize that standard controls can be designed for evaluating a number of parameters (e.g., RNA levels, polypeptide levels, specific cell types, etc.).

The term "expression" refers to the production of a gene product (e.g., a protein). The term "transient" when referring to expression means that the polynucleotide is not incorporated into the genome of the cell. The term "stable" when referring to expression means that the polynucleotide is incorporated into the genome of the cell, or that expression of the transgene is maintained using a positive selection marker (i.e., an exogenous gene expressed by the cell that has benefits under certain growth conditions).

The term "cytokine" refers to a general class of biomolecules that affect cells of the immune system. Exemplary cytokines include, but are not limited to, interferons and Interleukins (IL), particularly IL-2, IL-12, IL-15, IL-18, and IL-21. In a preferred embodiment, the cytokine is IL-2.

The term "cytokine which regulates the tumor microenvironment" is intended to mean a cytokine consisting of

The cells express and function as molecules that increase the anti-tumor response. Certain cytokines can suppress the response of the endogenous immune system to tumors and thus reduce the effectiveness of immunotherapy in treating cancer. Thus, the term also includes cytokine inhibitors that promote tumor growth, such as peptide inhibitors and/or ligands or receptors that bind to tumor growth promoting cytokines (e.g., ligand traps).

As used herein, the term "vector" refers to a non-chromosomal nucleic acid comprising an intact replicon, such that the vector can be replicated when placed in a permissive cell, e.g., by a transformation process. A vector may replicate in one cell type (such as a bacterium), but has limited or no ability to replicate in another cell (such as a mammalian cell). The vector may be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers, such as heterogeneous polylysine, oligopeptides of defined length, and polyethyleneimine, in some cases in liposomes; and the use of a ternary complex comprising a virus and polylysine-DNA. In one embodiment, the vector is a viral vector, e.g., an adenovirus. Viral vectors are well known in the art.

As used herein, the term "targeted" when referring to protein expression is intended to include, but is not limited to, directing a protein or polypeptide to an appropriate destination within or outside of a cell. Targeting is typically achieved by a signal or targeting peptide that is a stretch of amino acid residues in the polypeptide chain. These signal peptides may be located anywhere within the polypeptide sequence, but are typically located at the N-terminus. The polypeptide may also be engineered to have a signal peptide at the C-terminus. The signal peptide can direct the polypeptide to undergo extracellular cleavage, localization to the plasma membrane, golgi apparatus, endosomes, endoplasmic reticulum, and other cellular compartments. For example, a polypeptide having a particular amino acid sequence at its C-terminus (e.g., KDEL) is retained in or transported back into the ER lumen.

As used herein, the term "targeting" when referring to the targeting of a tumor refers to

The ability of cells to recognize and kill tumor cells (i.e., target cells). The term "targeted" in this context means, for example, a compound of formula (I)

The ability of a cell-expressed CAR to recognize and bind to a cell surface antigen expressed by a tumor.

The term "transfection" as used herein refers to the insertion of a nucleic acid into a cell. Any means of allowing nucleic acids to enter the cell may be used for transfection. The DNA and/or mRNA may be transfected into the cell. Preferably, the transfected cells express the gene product (i.e., protein) encoded by the nucleic acid.

Headings or subheadings may be used in the description for the convenience of the reader, but are not intended to affect the scope of the invention. In addition, some terms used in the present specification are defined more specifically below.

Detailed Description

Provided herein are engineered cells that are capable of,it uses a cytotoxic activated natural killer cell line (NK-92) as a basis to improve immunotherapy against cancer and tumors and/or increase homing (migration) to target targets. In some embodiments of the present invention, the,

the cells are engineered to express homing receptors known to direct lymphocytes to lymph nodes upon expression. In some embodiments of the present invention, the,

the cells are engineered to express secreted cytokines that regulate the tumor microenvironment or to block inhibitors of cytokines that regulate the tumor microenvironment.

The present disclosure provides the benefit of using a tetra-cistronic vector to insert multiple genes driven by a single high activity promoter to generate stable immunotherapeutic cell lines for clinical immunotherapy. The tetra-cistronic vector uses one or more methods (including the P2A peptide and IRES element) to string the four genes together under the control of a single promoter and to link the expression of the final element-in this case, a selective agent that requires expression to maintain cell survival and/or expansion.

In the proof of concept example, the four genes used to generate the modified gene expression profile in the therapeutic cell line were: CCR7, CD19 chimeric antigen receptor, high affinity variants of CD16, and endoplasmic reticulum-bound IL-2. ER-bound IL-2 is based, in addition to its ability to integrate it into it upon stimulation

In addition to the cytotoxic ability of the cell line of (a), also as a selective agent. The gene producing IL-2 is placed at the end of the tetra-cistronic vector, furthest away from the promoter, and therefore the most likely element to be lost should be a fragment of the gene construct (leading to negative selection and self-excision from the pool, since IL-2 is required for continued cell survival). However, if all elements were successfully integrated into the genome, cells would be selected by removing IL-2 from the medium, since only those areCells that have integrated their own source of IL-2 will survive. Since the IL-2 element is furthest from the promoter, this facilitates complete integration of the entire cassette for the four elements-as can be further verified by flow cytometry staining analysis of the other components (see examples).

The constructs described herein provide the advantage of reducing development time to generate new therapeutic cell lines, as well as reducing stress and adverse effects on cells from multiple rounds of genomic manipulation and subsequent selection. In addition, by placing the selection agent (in this case ER-IL-2) at the end of the construct, it is expected that the cell will have difficulty silencing any given component of the construct without causing starvation of IL-2 due to the nature of RNA transcription and processing. Thus, stable cell lines constructed in this manner should maintain expression of all components already contained in the vector, as long as they continue to produce their own IL-2.

To demonstrate proof of concept, four specific goals were addressed by 4 components as part of this tetra-cistronic construct. IL-2 acts as a selective agent and is

Known agonists of the cytotoxic effects of cells make these cells effective cancer therapeutics. The CCR7 element (C-C chemokine receptor type 7) is a chemokine receptor responsible for migrating immune cells expressing it in the direction of the chemokine gradient of the ligands CCL19 and CCL21 normally expressed in lymph nodes. In one embodiment, the CCR7 element contemplated herein may comprise a sequence identical to SEQ ID NO: 1(CCR7 sequence) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, CCL21 contemplated herein may comprise a sequence identical to SEQ ID NO: 2 (a CCL21 sequence) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In one embodiment, CCL19 contemplated herein may comprise a sequence identical to SEQ ID NO: 16(CCL19 sequence) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%A polynucleotide sequence of% identity, 96%, 97%, 98% or 99%.

CD19 CARs (chimeric antigen receptors) are constructs used to increase the cytotoxicity of cells as they encounter a differentiated population of CD19 on the cell surface they encounter, which is a differentiated population that is normally expressed on normal and malignant B cells, and have shown efficacy in directed immunotherapy trials against B-cell lymphomas. The high affinity CD16 receptor allows for modification

The cells recognize and respond to cells that have been recognized by IgG antibodies, such as those used as monoclonal antibodies in cancer treatment protocols, e.g., rituximab (Rituxumab) and Herceptin (Herceptin). When a CD16 receptor armed immune cell encounters a cell encapsulated in one of these antibodies, it triggers ADCC (antibody dependent cellular cytotoxicity) and attempts to destroy the cell. In fact, this allows cells that have been so armed to be used in combination therapy regimens with monoclonal antibodies against cancer neoantigens and the like. Although each of these components has its own utility, it is suggested that combining this particular combination will result in an effective therapy for B cell lymphomas that are capable of migrating to common sites of tumor outgrowth (lymph nodes), where the B cell antigen CD19 is recognized and CAR-mediated cytotoxicity is initiated, or working with monoclonal antibodies (such as rituximab) to avoid the possibility of antigen escape. It should be understood that this proof of concept example is non-limiting and can be modified using the methods described herein

Cells to express other homing receptors and/or CARs that target other antigens of interest in order to generate effective immunotherapeutic cell lines.

Modified has been produced as described herein

Cells containing the CCR7 expression cassetteFollowing electroporation of linearized gene constructs with removable selection cassettes of selection markers, stable long-term expression of CCR7 lymph node homing receptor driven by the elongation factor 1a (EF1a) promoter was achieved. One week after puromycin selection, followed by serial dilution cloning, a monoclonal cell line was established that maintained high levels of CCR7 expression. These NK cells overexpressing CCR7 were functionally responsive to the lymph node-associated chemokines CCL21 and CCL19 in migration/invasion assays. In one embodiment, the EF1a promoter contemplated herein can comprise a sequence identical to SEQ ID NO: 3(EF1a promoter sequence) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

In some exemplary embodiments, the chemokines and homing receptors contemplated herein may comprise a sequence identical to SEQ ID NO: 44(CCR7 amino acid sequence), or SEQ ID NO: 45(CCL19 amino acid sequence), or SEQ ID NO: 46(CCL21 amino acid sequence) or SEQ ID NO: 47(CXCR2 nucleotide sequence), or SEQ ID NO: 48(CXCR2 amino acid sequence), or SEQ ID NO: 49(CXCL14 nucleotide sequence), or SEQ ID NO: 50(CXCL14 amino acid sequence), or SEQ ID NO: 51(CD62L nucleotide sequence), or SEQ ID NO: 52(CD62L amino acid sequence), or SEQ ID NO: 53(IL-8 nucleotide sequence), or SEQ ID NO: 54(IL-8 amino acid sequence), or SEQ ID NO: 55(CXCL1 nucleotide sequence), or SEQ ID NO: 56(CXCL1 amino acid sequence) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

Through the activation of NFAT transcription factor and its nuclear translocation, it is confirmed that

Target engagement in cells that recognize susceptible cell lines. Target binding involving the FceRIg or CD3 zeta pathway (including ADCC or CAR-mediated target recognition) is sufficient to prevent binding of the target to the FceRIg or CD3 zeta pathway

Inducing NFAT activation in cells. This is achieved by inserting NFAT binding junctions flanking 3 termination regionsThe domain and a reporter cassette that drives the minimal promoter of firefly luciferase. Activation of NFAT via the CD3 ζ pathway followed by co-culture with SUP-B15(CD19+, but resistant to non-specific cytotoxicity) resulted in luciferase expression by electroporation of CD19CAR mRNA into the reporter cell line.

In one embodiment, the NFAT responsive element sequences contemplated herein (binding site for activating NFAT) may comprise a sequence that is identical to SEQ ID NO: 4, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

In one embodiment, the minimal promoter contemplated herein (downstream of 3 NFAT response elements) may comprise a sequence identical to SEQ ID NO: 5, or a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

In one embodiment, an intact NFAT response cassette (poly a + pause site, followed by 3 NFAT response elements, followed by a minimal promoter) contemplated herein may comprise a sequence identical to SEQ ID NO: 6, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

In one embodiment, the complete sequences contemplated herein for initial insertion (EF1a promoter, CCR7 gene with poly a, and LoxP flanking puromycin resistance gene driven by ubiquitin promoter, all packaged in the homology arm targeting the AAVS1 locus) may comprise a sequence identical to SEQ ID NO: 7, or a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

In one embodiment, the complete sequence contemplated herein for the second insertion (containing the NFAT response cassette driving CCL21+ polya and the FRT embedded blasticidin resistance gene driven by CMV) may comprise a sequence identical to SEQ ID NO: 8, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. SEQ ID NO: the 8 sequence may replace the LoxP flanking puromycin resistance cassette from the first insertion, i.e. the FRT sequence surrounding the blasticidin gene in this sequence, to allow for later removal or replacement of the cassette using similar Flp-FRT recombination.

The cell line was a human IL-2 dependent NK cell line established from Peripheral Blood Mononuclear Cells (PBMC) of a 50 year old male diagnosed with non-Hodgkin's lymphoma (Gong et al, Leukemia [ Leukemia ]]8：652-8(1994))。

The cells are characterized by expressing CD56 in the absence of CD3, CD8, and CD16^{Bright Light (LIGHT)}And CD 2. CD56^{Bright Light (LIGHT)}/CD16^{Negative of}The/low phenotype is typical of a small fraction of NK cells in peripheral blood, which have an immunomodulatory function as cytokine producers. Unlike normal NK cells, the cells are not,

lack of expression of most killer Cell inhibitor receptors (KIRs) (Maki et al, J Hematother Stem Cell Res. [ J.hematopoietic Stem Cell research ]]10: 369-83(2001)). Only KIR2DL4 was detected on the surface of NK-92, a KIR receptor with activating function and inhibitory potential expressed by all NK cells. KIR2DL4 is thought to mediate inhibition by binding to HLA allele G (mock, Cancer immunol]65(4)：485-92(2015))。

The primary route of cytotoxic killing of cells is through the perforin/esterase pathway;

expression of high levels of perforin and granzyme B (Maki et al, J Hematother Stem Cell Res. [ J.Hematother Stem Cell Res. ]]10：369-83(2001))。

The cells have a very broad cytotoxicity range and are active against cell lines derived from hematological malignancies and solid tumors (Klingemann, Blood [ Blood ] Blood],87(11): 4913-4 (1996); swift, Haematologica [ hematology]97(7): 1020-8 (2012); yan et al, Clin Cancer Res [ clinical Cancer research ]]4: 2859-68(1998)). Safety assessments in Severe Combined Immunodeficiency (SCID) mice did not show any cross-correlation with

Treatment-related effects, such as acute toxicity or long-term carcinogenicity (Tam et al, J Hemalt [ J.Hemaother. [ J.Hematotherapy J. ]]8: 281-90(1999), Yan et al, Clin Cancer Res [ clinical Cancer research]4: 2859-68(1998)). Will be provided with

Cell administration to mice challenged with human leukemia cells or to human melanoma mouse models resulted in improved survival and inhibition of tumor growth, including complete remission in some mouse tumors (Tam et al, J hemarther [ J.Hemato.C. ]]8: 281-90(1999), Yan et al, Clin Cancer Res [ clinical Cancer research]4: 2859-68(1998)). Phase I clinical trials have demonstrated their safety. In WO 1998/49268 and U.S. patent application publication No. 2002-0068044

Characterization of cell lines, both of which are incorporated herein by reference in their entirety.

Optionally modified

The cells may also express the Fc receptor CD16. As used herein, the term "Fc receptor" refers to a protein found on the surface of certain cells (e.g., natural killer cells) that contributes to the protective function of immune cells by binding to the portion of an antibody referred to as the Fc region. Binding of the Fc region of an antibody to the Fc receptor (FcR) of a cellThe phagocytosis or cytotoxic activity of cells is stimulated by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcR is classified by the type of antibody it recognizes. For example, Fc-gamma receptors (Fc γ R) bind IgG class antibodies. FC γ RIII-a (also known as CD16) is a low affinity FC receptor that binds to IgG antibodies and activates ADCC. FC γ RIII-A is commonly found on NK cells. A representative amino acid sequence encoding CD16 is set forth in SEQ ID NO: shown at 12. Representative polynucleotide sequences encoding CD16 are set forth in SEQ ID NO: shown at 13. The complete sequence of CD16 can be found in the SwissProt database as per entry P08637.

In some embodiments, the CD16 receptor comprises a phenylalanine (F) -valine (V) substitution at amino acid position 158(F158V) in the IgG binding domain of the mature CD16 receptor (corresponding to Val at position 176 of the full-length protein) that affects antibody-dependent cellular cytotoxicity (ADCC) function of NK cells. The CD16158V variant binds with higher affinity to human IgG1 and IgG3 than the 158F variant.

Optionally modified

The cell comprises a nucleotide sequence identical to SEQ ID NO: 13, or a nucleic acid sequence having 70%, 80%, 90% or 95% identity thereto. Optionally modified

The cell comprises a nucleotide sequence identical to SEQ ID NO: 13, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. Optionally modified

The cell comprises a nucleotide sequence identical to SEQ ID NO: 12 (having a valine at position 176 of the full-length polypeptide) with 70%, 80%, 90% or 95% identity. Optionally modified

The cell comprises a nucleotide sequence identical to SEQ ID NO: 12 has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%Polypeptides of 98% or 99% identity.

Cytotoxicity of cells depends on the presence of cytokines, e.g., interleukin 2 (IL-2). Thus, optionally, modified

The cells are further modified to express at least one cytokine. Optionally, the at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21, or a variant thereof. Optionally, the at least one cytokine is IL-2, IL-15, or a combination thereof. Optionally, IL-2 and/or IL-15 is expressed with a signal sequence that directs the cytokine to the endoplasmic reticulum. Directing IL-2 to the endoplasmic reticulum allows IL-2 to be expressed at levels sufficient for autocrine activation without releasing large amounts of IL-2 extracellularly. See Konstantinidis et al, "Targeting IL-2 to the end plastic suspensions derivatives and growth stimulation to

cells [ targeting IL-2 to the endoplasmic reticulum limits autocrine growth stimulation to

In cells]"Exp hematosol" [ experimental hematology ]]2 months in 2005; 33(2): 159-64. Representative nucleic acids encoding IL-2 are set forth in SEQ ID NO: 14, and a representative polypeptide of IL-2 is set forth in SEQ ID NO: shown at 15.

Optionally modified

The cell comprises a nucleotide sequence identical to SEQ ID NO: 14 nucleic acid sequence encoding IL-2 having 70%, 80%, 90% or 95% identity. Optionally modified

The cell comprises a nucleotide sequence identical to SEQ ID NO: 14 has 90%, 91%, 92%, 93%,A nucleic acid sequence of 94%, 95%, 96%, 97%, 98% or 99% identity. Optionally modified

The cell comprises a nucleotide sequence identical to SEQ ID NO: 15 an IL-2 polypeptide having 70%, 80%, 90% or 95% identity. Optionally modified

The cell comprises a nucleotide sequence identical to SEQ ID NO: 15, an IL-2 polypeptide having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Modified provided

The cells are advantageously able to be maintained in the absence of IL-2 without secreting IL-2 in amounts that cause clinical side effects.

In one aspect, the subject matter of the invention includes modified polypeptides capable of modulating the tumor microenvironment

A cell. Modified

The cell preferably comprises a tetra-cistronic vector comprising one or more nucleic acids encoding: i) IL-12 or TGF- β trap, ii) an Antigen Binding Protein (ABP) or Chimeric Antigen Receptor (CAR) that specifically binds to a target antigen, iii) an Fc receptor, such as CD16 or CD16-158V, and/or iv) a cytokine, such as erll-2 or erll-15, wherein the nucleic acid sequence is operably linked to a promoter. In one embodiment, the tetra-cistronic vectors contemplated herein are illustrated in fig. 21, fig. 30, and fig. 31, respectively. IL-12 contemplated herein may comprise an amino acid sequence identical to SEQ ID NO: 57(p35 nucleotide sequence) or SEQ ID NO: 59(p40 nucleotide sequence) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. IL-12 contemplated herein may also comprise a sequence identical to SEQ ID NO: 58(p35 amino acid sequence)Column, isoform 1 precursor) or SEQ ID NO: 60(p40 amino acid sequence, precursor) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity amino acid sequence.

In an exemplary embodiment, the IL-12 single-chain p40_ p35 sequence in the IL-2/PD-L1 tetra-cistronic vector may comprise a sequence identical to SEQ ID NO: 61, or may comprise a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 62, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

TGF- β traps contemplated herein may comprise a sequence identical to SEQ ID NO: 63(TGFBRII extracellular domain) or SEQ ID NO: 65(TGFb trap sequence) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. TGF- β traps contemplated herein may further comprise a sequence identical to SEQ ID NO: 64(TGFBRII extracellular domain) or SEQ ID NO: 66(TGFb trap sequence) has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. Other suitable TGF- β traps include those described in mol. can. ther. [ molecular cancer therapy ]2012, vol 11 (7), 1477-.

In addition, the nucleic acid constructs of the inventive subject matter may also comprise sequences encoding 2A peptides, such as T2A, P2A, E2A, or F2A peptides, in order to produce equimolar levels of the polypeptides encoded by the same mRNA. The E2A peptides contemplated herein may comprise an amino acid sequence identical to SEQ ID NO: 17, or a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The T2A peptides contemplated herein may comprise a sequence identical to SEQ ID NO: 18, or a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

In an illustrative, non-limiting example, a plasmid disclosed herein can comprise a nucleotide sequence identical to SEQ ID NO: 19 (5' homology arm of AAVS 1), SEQ ID NO: 20(EF1a promoter), SEQ ID NO: 21(T7 promoter), SEQ ID NO: 22(CCR7 cDNA), SEQ ID NO: 23(P2A element), SEQ ID NO: 24(IgHC leader sequence), SEQ ID NO: 25(CD19 CAR minus signal peptide), SEQ ID NO: 26 (high affinity CD16), SEQ ID NO: 27(IRES), SEQ ID NO: 28(SC40 polya), SEQ ID NO: 29 (3' homology arm of AAVS 1) and/or SEQ ID NO: 30 (homology arms of AAVS 1) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

Chimeric antigen receptors

Optionally, modifying

The cells are further engineered to express a Chimeric Antigen Receptor (CAR) on the cell surface. Optionally, the CAR is specific for a tumor-specific antigen. As a non-limiting example, tumor specific antigens are described in US 2013/0189268, WO 1999024566 a1, US 7098008 and WO 2000020460 a1, each of which is incorporated herein by reference in its entirety. Tumor specific antigens include, but are not limited to, NKG2D, CS1, GD2, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, MAGE, CAGE, BAGE, HAGE, LAGE, PAGE, NY-SEO-1, GAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, AFP, CEA, CTAG1B, CD19, CD33, B7-H4, CD20 and 41 BB. The CAR may be designed as described, for example, in patent publications nos. WO 2014039523, US 20140242701, US 20140274909, US 20130280285, and WO 2014099671, each of which is incorporated herein by reference in its entirety. Optionally, the CAR is a CD19CAR, a CD33 CAR, or a CSPG-4 CAR. In an illustrative, non-limiting example, CD19CAR _ CD3a may comprise a sequence identical to SEQ ID NO: 41, or a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

Homing receptor

Provided herein are modified nucleic acids comprising homing receptor-encoding nucleic acids

A cell. In some embodiments, the homing receptor is operably linked to a promoter. In some embodiments, the homing receptor is a G protein-coupled receptor. In some embodiments, the homing receptor is a receptor selected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC, or CXCL14 receptors. In some embodiments, the nucleic acid encoding CCR7 is identical to SEQ ID NO: 1 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. Optionally, the homing receptor is modified

Expressed on the cell surface of the cell. Optionally modified

The cell further comprises a CAR. Optionally, the CAR is CD 19. Optionally modified

The cell further comprises an Fc receptor. Optionally, the Fc receptor is CD16. Optionally modified

The cells further comprise a cytokine, such as IL-2. In some embodiments, the IL-2 polypeptide can have an amino acid sequence identical to SEQ ID NO: 42, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In some embodiments, the IL-2 polypeptide can have an amino acid sequence identical to SEQ ID NO: 43 are sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

Expression vector

Provided herein are expression vectors comprising a nucleic acid operably linked to a promoter. The nucleic acids of the vector encoding the different elements may each be operably linked to the same or different promoters. Exemplary promoters include, but are not limited to, the CMV promoter, the ubiquitin promoter, the PGK promoter, and also the EF1 promoter. Optionally, provided herein are expression vectors comprising a polynucleotide having the sequence of SEQ ID NO: 1 or a nucleic acid corresponding to SEQ ID NO: 1, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. Optionally, the nucleic acid is operably linked to a promoter. Optionally, the promoter is selected from the group consisting of SEQ ID NO: 3. 9, 10 or 11 or a variant of SEQ ID NO: 3. 9, 10 or 11, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Optionally, the nucleic acid is operably linked to a promoter. Optionally, the promoter comprises SEQ ID NO: 4 and/or SEQ ID NO: 5. optionally, the promoter comprises SEQ ID NO: 6 or a sequence identical to SEQ ID NO: 6, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

In some embodiments, provided expression vectors comprise SEQ ID NO: 1 or a sequence identical to SEQ ID NO: 1, a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; SEQ ID NO: 25(CD19 CAR) or a variant of SEQ ID NO: 13(CD16158V), or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14 (erll-2 nucleotide sequence) a nucleic acid or polypeptide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; and/or SEQ ID NO: 15 (erll-2 amino acid sequence), or a sequence identical to SEQ ID NO: 14, a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In some embodiments, provided expression vectors comprise SEQ ID NO: 47(CXCR2) or a variant of SEQ ID NO: 47, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; SEQ ID NO: 25(CD19 CAR) or a variant of SEQ ID NO: 12, a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; and SEQ ID NO: 13(CD16158V), and/or a variant of SEQ ID NO: 14, a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; and/or SEQ ID NO: 15 (erll-2), or a variant of SEQ ID NO: 14, a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. Suitable expression vectors are known in the art and may be used. In a further aspect, the recombinant nucleic acid comprises a segment encoding erll-15, and the nucleic acid encoding erll-15 is identical to SEQ ID NO: 67 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Optionally, the expression vector is a plasmid.

The expression analysis of PD-L1 CAR and CD-16 in PD-L1(TGF β -trap) t-haNK cells is illustrated in FIG. 22. Furthermore, as shown in FIG. 23, TGF-beta trap was secreted into the culture supernatant of TGF-beta trap/PD-L1 t-hanK cells. Similarly, FIG. 30 right column shows IL-12 viral transduction

IL-12 secretion from cell lines.

Preparation of modified

Method of cell

Provided herein are methods of making modified nucleic acid molecules comprising the nucleic acid molecules described herein

Methods of making cells. These methods include transformation with an expression vector

A cell, the expression vector comprising a nucleic acid described herein operably linked to a promoter.

As used herein, the terms promoter, promoter element, and regulatory sequence refer to a polynucleotide that regulates expression of a selected polynucleotide sequence operably linked to a promoter and effects expression of the selected polynucleotide sequence in a cell. In some embodiments, the promoter element is or comprises an untranslated region (UTR) at a 5' position of the coding sequence. The 5' UTR forms part of the mRNA transcript and is therefore also an integral part of protein expression in eukaryotes. After transcription, the 5' UTR can regulate protein expression at both the transcriptional and translational levels. Promoters controlling transcription from vectors in mammalian host cells can be obtained from a variety of sources, for example, viral, such as polyoma, simian virus 40(SV40), adenovirus, retrovirus, hepatitis B virus, and cytomegalovirus genomes (e.g., SEQ ID NO: 11), or from heterologous mammalian promoters, such as the beta actin promoter, eukaryotic translation elongation factor 1 alpha 1(EF1 alpha) promoter (e.g., SEQ ID NO: 3), phosphoglycerate kinase (PGK) promoter (e.g., SEQ ID NO: 10), and ubiquitin promoter (e.g., SEQ ID NO: 9). The promoters provided herein are substantially identical to SEQ ID NO: 3. 9, 10 or 11 have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

As used herein, the phrase selection marker refers to a nucleotide sequence (e.g., a gene) that encodes a product (polypeptide) that allows for selection, or to the gene product (e.g., polypeptide) itself. The term selection marker is used herein as it is generally understood in the art and refers to a marker whose presence within a cell or organism confers a significant growth or survival advantage or disadvantage to that cell or organism under certain defined culture conditions (selective conditions). The phrase selection agent, as used herein, refers to an agent that introduces a selection pressure on a cell or population of cells to favor or counteract the cell or population of cells bearing a selection marker. For example, the selection agent is an antibiotic and the selection marker is an antibiotic resistance gene. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin analog G418, hygromycin and puromycin.

Nucleic acid as used herein refers to deoxyribonucleotides or ribonucleotides and polymers and complements thereof. The term includes deoxyribonucleotides or ribonucleotides in either single-or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2-O-methyl ribonucleotide, Peptide Nucleic Acid (PNA). Unless otherwise indicated, conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences of a nucleic acid sequence may be used in place of the particular nucleic acid sequence recited herein. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. [ Nucleic Acid research ] 19: 5081 (1991); Ohtsuka et al, J.biol.chem. [ J.Biol. ] 260: 2605-. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

A nucleic acid is operably linked when it is in a functional relationship with another nucleic acid sequence. For example, a DNA encoding a presequence or secretory leader is operably linked to a DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or, if the ribosome binding site is positioned so as to facilitate translation, it is operably linked to a coding sequence. Generally, operably linked means that the DNA sequences being linked are in close proximity to each other and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers need not be contiguous. For example, a nucleic acid sequence operably linked to a second nucleic acid sequence is covalently linked, directly or indirectly, to such second sequence, but any effective three-dimensional association is acceptable. A single nucleic acid sequence may be operably linked to multiple other sequences. For example, a single promoter may direct transcription of multiple RNA species. Ligation may be accomplished by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

Method of treatment

Described herein are methods of treating cancer or a tumor in a subject. As used herein, the term "cancer" refers to all types of cancers, neoplasms or malignancies found in mammals, including leukemias, carcinomas and sarcomas. Exemplary cancers include brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, gastric cancer, uterine cancer, and medulloblastoma. Other examples include hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, primary brain tumor, cancer, malignant pancreatic islet tumor, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, endocrine and exocrine pancreatic neoplasms, and prostate cancer.

As used herein, the terms "metastasis," "metastatic," and "metastatic cancer" are used interchangeably and refer to the spread of a proliferative disease or disorder, such as cancer, from one organ or another non-adjacent organ or body part. Cancer occurs at a site of origin, such as breast cancer, which is referred to as a primary tumor, such as primary breast cancer. Some cancer cells in the primary tumor or site of origin acquire the ability to penetrate and infiltrate surrounding normal tissue in a localized area, and/or penetrate the wall of the lymphatic or vascular system through which the circulation to other sites and tissues in the body occurs. The second clinically detectable tumor formed by the cancer cells of the primary tumor is called a metastatic tumor or a secondary tumor. When cancer cells metastasize, it is presumed that metastatic tumors and their cells are similar to the original tumor. Thus, if lung cancer metastasizes to the breast, secondary tumors at the breast site consist of abnormal lung cells rather than abnormal breast cells. Secondary tumors in the breast are called metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrase non-metastatic cancer or a subject with a non-metastatic cancer refers to a disease in which the subject has a primary tumor but does not have one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject who has a primary lung tumor or has a history of a primary lung tumor and one or more secondary tumors at a second location or locations (e.g., in the breast).

As used herein, "treating" a condition, a disease or disorder and a condition, a disease or disorder-associated symptom or condition, a disease or disorder and a condition, disease or disorder-associated symptom "treatment" refers to a method of obtaining a beneficial or desired result, including a clinical result. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of a condition, disorder, or disease, stabilization of the state of a condition, disorder, or disease, prevention of progression of a condition, disorder, or disease, prevention of spread of a condition, disorder, or disease, delay or slowing of progression of a condition, disorder, or disease, delay or slowing of onset of a condition, disorder, or disease, amelioration or palliation of a condition, disorder, or disease state, and partial or complete remission. "treating" may also mean prolonging the survival of a subject beyond that expected in the absence of treatment. "treating" may also mean temporarily inhibiting the progression of the condition, disorder, or disease, temporarily slowing the progression of the condition, disorder, or disease, but in some cases it involves permanently halting the progression of the condition, disorder, or disease. As used herein, the term treatment refers to a method of reducing the effect of one or more symptoms of a disease or condition characterized by protease expression or symptoms of a disease or condition characterized by protease expression. Thus, in the disclosed methods, treatment may refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an identified disease, condition, or symptom of the disease or condition. For example, a method of treating a disease is considered treatment if one or more symptoms of the disease are reduced by 10% in a subject as compared to a control. Thus, the amount of reduction compared to the native or control level can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage between 10% and 100%. It is to be understood that treatment does not necessarily refer to a cure or complete ablation of a disease, condition, or symptom of the disease or condition. Further, as used herein, reference to reduction, alleviation, or inhibition includes a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, as compared to a control level, and such terms may include, but do not necessarily include, complete elimination.

The terms subject, patient, individual, etc. are not intended to be limiting and are generally interchangeable. That is, an individual described as a patient does not necessarily have a given disease, but may simply be seeking medical advice. As used throughout, a subject may be a vertebrate, more specifically a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, and any other animal. The term does not specify a particular age or gender. Thus, it is intended to cover adult and newborn subjects, whether male or female. As used herein, patient, individual, and subject may be used interchangeably, and these terms are not intended to be limiting. That is, an individual described as a patient does not necessarily have a given disease, but may simply be seeking medical advice. The term patient or subject includes human and veterinary subjects.

As used herein, "administering" refers to providing, contacting, and/or delivering a compound or compounds by any suitable route to achieve a desired effect. Administration can include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection), transdermal, topical, buccal, rectal, vaginal, nasal, ocular, inhalation, and implantation. Optionally, the step of (a) is carried out,

the cells are administered parenterally. Optionally, optionally，

Cells were administered intravenously. Optionally, the step of (a) is carried out,

the cells are administered peritumorally.

Thus, provided herein are methods of reducing cancer metastasis in a subject, comprising administering to the subject a therapeutically effective amount of a modified as described herein

A cell, thereby reducing cancer metastasis in the subject. Also provided are methods of treating cancer in a subject, comprising selecting a subject having cancer and administering to the subject a therapeutically effective amount of a modified antibody described herein

A step of treating a cancer in a subject, wherein the administration is. Optionally, the methods further comprise administering to the subject an additional therapeutic agent.

In some embodiments, the methods further comprise administering to the subject a therapeutically effective amount of a modified as described herein

A cell, wherein the administration treats cancer or reduces tumor size in the subject. In some embodiments, the methods comprise administering the modified to a subject

Cells comprising nucleic acids encoding: i) IL-12 or TGFb trap, ii) ABP or CAR that specifically binds to a target antigen, iii) Fc receptors such as CD16 or CD16-158V, and/or iv) cytokines such as erll-2 or erll-15. In some embodiments, the methods comprise administering the modified to a subject

Cells comprising nucleic acids encoding: i) a homing receptor, ii) an ABP or CAR that specifically binds to a target antigen, iii) an Fc receptor such as CD16 or CD16-158V, and/or iv) a cytokine such as erll-2 or erll-15.

The cytotoxicity of TGF β trap/PD-L1 on K562 target cells is illustrated in figure 24. Also shown in FIG. 25 is a view directed to SUP-B15^PD-L1+CAR killing of target cells, while CAR killing against MDA-MB 231 target cells is shown in figure 26. In FIG. 27 is illustrated TGF beta trap/PD-L1 vs. SUP-B15^CD19-CD20+ADCC of (1). FIG. 28 illustrates that TGF β/SMAD luciferase reports that HEK293 cells are induced by TGF β. Secreted TGF β -trap chelated TGF β and inhibited luciferase expression in the HEK293T reporter assay are shown in figure 29. Reporter cells were treated with 1ng/mL TGF β 1 for 19 hours. In other options, preferred CAR molecules specifically bind PD-L1 and may have an amino acid sequence that is identical to SEQ ID NO: 69 (which may be encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 68, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

Cells can be administered to a subject by a variety of routes. For example, the composition may be administered by infusion (e.g., intravenous infusion) over a period of time

The cells are administered to a subject. Typically, for a single dose

Cells, for a period of 5 to 130 minutes. Optionally, the time period is between 90 and 120 minutes. Optionally, the time period is between 15 and 30 minutes.

The cells and optionally other anti-cancer agents may be administered once or they may be administered multiple times during therapy to a patient with cancer, for example once every 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or once every 1, 2, 3, 4, 5, 6, or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8,9, 10 weeks or more, or any range between any two of these numbers (including endpoints). Thus, for example, the subject may be administered daily

The cells are administered once for 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. Optionally, administration is carried out once daily for a period of two days

A cell. Not used one or more hours, days or weeks after the period

And (4) cell therapy. As used herein, the term "cycle" refers to a treatment that is repeated on a regular schedule with a rest period in between (e.g., no treatment or treatment with other agents). For example, administration of a one week treatment followed by a two week rest is one treatment cycle. Such treatment cycles may be repeated one or more times. Thus, administration can be in 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more cycles

A cell.

Can be administered to a subject in absolute numbers of cells

Cells, for example, about 1000 cells per injection may be administered to the subjectUp to about 100 hundred million cells per injection, such as about, at least about, or up to about 1x 10 per injection¹⁰、1×10⁹、1×10⁸、1×10⁷、5×10⁷、1×10⁶、5×10⁶、1×10⁵、5×10⁵、1×10⁴、5×10⁴、1×10³、5×10³One (and so on)

A cell, or any range between any two of these numbers (inclusive). Optionally, 1 × 10⁸To 1X 10¹⁰The individual cells are administered to a subject. Optionally, the cells are administered one or more times per week for one or more weeks. Optionally, the cells are administered once or twice a week for 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more weeks.

Optionally, one can inject at about 1000 cells/injection/m²Up to about 100 hundred million cells per injection/m²Administration to a subject, such as about, at least about, or at most about 1X 10 per injection¹⁰Per m²、1×10⁹Per m²、1×10⁸Per m²、1×10⁷Per m²、5×10⁷Per m²、1×10⁶Per m²、5×10⁶Per m²、1×10⁵Per m²、5×10⁵Per m²、1×10⁴Per m²、5×10⁴Per m²、1×10³Per m²、5×10³Per m²(and so on)

A cell, or any range between any two of these numbers (inclusive). Optionally, every m to the subject²Application of 1X 10³To 1X 10¹⁰An

A cell. Optionally, every m to the subject²Application of 2X 10⁹An

A cell.

Optionally, such individuals may be administered the relative number of cells

Cells, for example, from about 1000 cells per kilogram of individual to up to about 100 hundred million cells per kilogram of individual, such as about, at least about, or up to about 1x 10¹⁰、1×10⁹、1×10⁸、1×10⁷、5×10⁷、1×10⁶、5×10⁶、1×10⁵、5×10⁵、1×10⁴、5×10⁴、1×10³、5×10³(and so on) one

Cells per kilogram of individual, or any range between any two of these numbers (inclusive).

Optionally, the total dose may be in terms of body surface area m²Performing calculations, including per m²About 1X 10¹¹1, 1 × 10¹⁰1, 1 × 10⁹1, 1 × 10^{8 are provided with}、1×10⁷Or any range between any two of these numbers (including endpoints). Optionally, about 10 to about 30 million of the total weight of the composition is administered to the patient

A cell. Optionally, each dose is injected

The amount of cells can be calculated from the body surface area m2, including per m²Is 1 × 10¹¹1, 1 × 10¹⁰1, 1 × 10⁹1, 1 × 10⁸1, 1 × 10⁷1, 1 × 10⁶1, 1 × 10⁵1, 1 × 10⁴1, 1 × 10³And (4) respectively.

Optionally, to contain

Administration in the form of a composition of cells and culture medium (such as human serum or its equivalent)

A cell. Optionally, the culture medium comprises human serum albumin. Optionally, the culture medium comprises human plasma. Optionally, the culture medium comprises about 1% to about 15% human serum or human serum equivalent. Optionally, the culture medium comprises about 1% to about 10% human serum or a human serum equivalent. Optionally, the culture medium comprises about 1% to about 5% human serum or a human serum equivalent. Optionally, the culture medium comprises about 2.5% human serum or a human serum equivalent. Optionally, the serum is human AB serum. Optionally, a serum replacement acceptable for use in human therapeutics is used in place of human serum. Such serum replacement may be known in the art. Optionally, to contain

Administration of cells in the form of a composition of cells and an isotonic liquid solution that supports cell viability

A cell. Optionally, administration is in the form of a composition reconstituted from a cryopreserved sample

A cell.

According to the methods provided herein, an effective amount of one or more agents provided herein is administered to a subject. The terms effective amount and effective dose are used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiological response (e.g., to reduce inflammation). One skilled in the art can empirically determine the effective amount and schedule of administration of the agent. Dosage ranges administered are those that are large enough to produce a desired effect in which one or more symptoms of a disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause serious adverse side effects such as unwanted cross-reactions, allergic reactions, and the like. In general, the dosage will vary with age, condition, sex, type of disease, extent of disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by one skilled in the art. The dosage may be adjusted by the individual physician if any contraindications occur. The dosage may vary, and may be administered once or more times per day for one or more days. Guidelines for appropriate dosages can be found in the literature for a given class of pharmaceutical products. For example, for a given parameter, an effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy may also be expressed as "how many times" an increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect relative to a control. The exact Dosage and formulation will depend on The purpose of The treatment and will be determined by those skilled in The Art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Vol. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding [ field, Science and Technology of drug complexation ] (1999); Remington: The Science and Practice of Pharmacy [ Remington: Pharmaceutical Science and Practice ], 22 nd edition, Gennaro's edition (2012), and Pickar, Pharmaceutical procedures [ dose calculation ] (1999)).

Pharmaceutically acceptable compositions may comprise a variety of carriers and excipients. Various aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of unwanted substances. Suitable carriers and formulations thereof are described in Remington: the Science and Practice of Pharmacy [ Remington: pharmaceutical science and practice ], 22 nd edition, edited by Loyd v. By pharmaceutically acceptable carrier is meant a material that is not biologically or otherwise undesirable, i.e., administration of the material to a subject does not cause undesirable biological effects or interact in a deleterious manner with other components contained in the pharmaceutical composition. If administered to a subject, the carrier is optionally selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject. As used herein, the term pharmaceutically acceptable is used synonymously with physiologically acceptable and pharmacologically acceptable. Pharmaceutical compositions typically comprise agents for buffering and preservation in storage, and may include buffers and carriers for appropriate delivery depending on the route of administration.

These compositions may contain acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of cells in these formulations and/or other agents can vary, and will be selected primarily according to fluid volume, viscosity, body weight, etc., according to the particular mode of administration selected and the needs of the subject.

Combination therapy

Optionally, mixing

The cells are administered to the subject with one or more other treatments for the cancer being treated. Without being bound by theory, it is believed

Treatment of a subject with cells in combination with another cancer therapy will result in

Cellular and replacement therapies provide the endogenous immune system with the opportunity to clear cancers that heretofore overwhelm such endogenous effects. Optionally, the two or more other treatments for the cancer being treated include, for example, antibodies, bispecific adaptors, radiation, chemotherapeutic agents, stem cell transplantation, or hormone therapy.

Optionally, the antibody is conjugated to

The cells are administered to the patient together. Optionally, the step of (a) is carried out,

the cells and antibody are administered to the subject together, e.g., in the same formulation; for example, in separate formulations, simultaneously separately administered to the subject; or may be administered separately, e.g., on a different dosing schedule or at different times of the day. When administered alone, the antibody may be administered by any suitable route, such as intravenously or orally.

Optionally, the antibodies can be used to target cancer cells or cells expressing cancer-associated markers. Many antibodies have been approved for use alone in the treatment of cancer.

The provided methods may further be combined with other tumor therapies such as radiation therapy, surgery, hormonal therapy, and/or immunotherapy. Thus, the provided methods may further comprise administering to the subject one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, analgesics, anesthetics, stimulants, corticosteroids, anticholinergics, anticholinesterases, anticonvulsants, antineoplastics, allosteric inhibitors, anabolic steroids, antirheumatics, psychotherapeutics, nerve blockers, anti-inflammatory agents, anthelmintics, antibiotics, anticoagulants, antifungals, antihistamines, antimuscarinics, antiprotozoals, antivirals, dopaminergic agents, hematologic agents, immunological agents, muscarinic agents, protease inhibitors, vitamins, growth factors, and hormones. The choice of agent and dosage can be readily determined by one skilled in the art based on the given disease being treated. Optionally, the additional therapeutic agent is octreotide acetate (octreotide acetate), interferon, pembrolizumab (pembrolizumab), glucopyranosyl lipid a, carboplatin (carboplatin), etoposide (etoposide), or any combination thereof.

Optionally, the additional therapeutic agent is a chemotherapeutic agent. A chemotherapeutic treatment regimen may include administering one or a combination of chemotherapeutic agents to a subject. Chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, taxanes, epothilones (epothilones), histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, monoclonal antibodies, nucleotide analogs and precursor analogs, peptide antibiotics, platinum-based compounds, retinoids, vinca alkaloids, and derivatives thereof. Optionally, the chemotherapeutic agent is carboplatin.

The combination of agents or compositions can be administered concomitantly (e.g., as a mixture), separately but simultaneously (e.g., via separate intravenous lines) or sequentially (e.g., first administering one agent and then administering a second agent). Thus, the term combination is used to refer to the concomitant, simultaneous or sequential administration of two or more agents or compositions. The course of treatment is preferably determined on an individual basis based on the particular characteristics of the subject and the type of treatment selected. Treatments, such as those disclosed herein, can be administered to a subject once a day, twice a day, biweekly, monthly, or on any applicable basis where the treatment is effective. The treatment can be administered alone or in combination with any other treatment disclosed herein or known in the art. Additional treatments may be administered at different times or on a completely different treatment schedule than the first therapy (e.g., the first therapy may be once daily and the additional treatments once weekly).

Reagent kit

Provided herein are compositions comprising the modified polymers described herein

A kit of cells. In some embodiments, the kit comprises a modified

Cells comprising operably linked to a promoter a nucleic acid encoding: i) a homing receptor, ii) an ABP or CAR that specifically binds to a target antigen, iii) an Fc receptor (such as CD16 or CD16-158V), and/or iv) a cytokine (such as erll-2). Optionally, one or more proteins encoded by the nucleic acid sequence are modified

Expressed on the cell surface of the cell. In some embodiments, the kit comprises a modified

A cell comprising a nucleic acid encoding a C-C chemokine receptor type 7 (CCR7), CXCR2, or CXCL14 receptor operably linked to a promoter. Optionally, the nucleic acid encoding CCR7 is identical to SEQ ID NO: 1 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. Optionally, the homing receptor is modified

Expressed on the cell surface of the cell. Optionally, the promoter comprises one or more NFAT-binding elements and a minimal promoter. Optionally, the promoter is identical to SEQ ID NO: 6 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. Optionally, one or more proteins encoded by the nucleic acid sequence are modified

Expressed on the cell surface of the cell.

Optionally, the modified is provided in a composition comprising a pharmaceutically acceptable excipient

A cell. Optionally, the kit may contain other compounds, such as therapeutically active compounds or compounds to be modified in the administration

A drug administered before, simultaneously with or after the cells. Optionally, instructions for use of the kit will include directions for using the kit components in the treatment of cancer. The instructions may further contain information on how to prepare the antibody (e.g., in the case of a lyophilized protein, diluted or reconstituted) and

information of cells (e.g., thawed and/or cultured). The instructions may further include guidelines for the dosage and frequency of administration.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are disclosed for the products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each individual and collective combination and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, unless explicitly stated to the contrary, if a method is disclosed and discussed and many modifications are possible to many molecules (including the method), then each and every combination and permutation of the method and the possible modifications are specifically contemplated. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of using the disclosed compositions. Thus, if multiple additional steps can be performed, it is understood that each of these additional steps can be performed with any specific method step or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

The publications cited herein and the materials cited therein are hereby incorporated by reference in their entirety.

The following examples are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims.

Examples of the invention

Example 1. modified NK cell line expressing the cytokine CCR7 that modulates the tumor microenvironment.

Using the NEON transfection system (Thermo Fisher Scientific, Waltham, Mass.), the plasmid pair was linearized with pNKAT-CCR7-LP3 (FIG. 1)

Subjecting the cells to electroporation to produce modified cells

A cell. After 1 week of puromycin selection, the resulting polyclonal population was tested for CCR7 expression and monoclonal cell lines were obtained by serial dilution in growth medium supplemented with 5% human serum and IL-2. Modified

The cells contained the EF1 alpha promoter, the CCR7 gene with a poly A tail, and the LoxP flanking puromycin resistance gene driven by the ubiquitin promoter, all of which were packaged in the homology arm targeting the AAVS1 locus (SEQ ID NO: 7).

To verify that expression of CCR7 did not affect

Cells, assay

Expression of cellular markers. The results are shown in FIG. 3. All of the data in FIG. 3 was generated using the Intellicy iQue scanner plus. Cells were incubated with APC-conjugated antibodies against the phenotypic markers or the appropriate isotype used as a negative control for 30min at 4 ℃. Cells were then washed in PBS + 1% BSA, pelleted, and resuspended in 30uL PBS + 1% BSA. The reading of the readout (readout) was then gated as shown in the upper left quadrant to eliminate cellular debris from the reading (reading), and then the percentage of cells above the fluorescence threshold shown in the upper right quadrant was displayed as two separate heat maps, showing the percentage above the "positive" threshold and the "strong positive" threshold in the lower left and lower right quadrants, respectively. Figure 3 shows that driving expression of CCR7 did not have a meaningful effect on the major phenotypic markers associated with our cell lines. Specifically, CCR7 expression does not appear to affect CD54, NKp30 or NKG2D expression.

To determine modified

Cytotoxicity of cells, effector cells: (A)

Cells and modified

Cell clones) were serially diluted in 96-well V-bottom plates, with 10 thousand effector cells remaining in the highest concentration well and 7 additional 2-fold dilutions in 8 rows of the plate. Stained target cells (K562 (fig. 4), HL-60 (fig. 5)) were then seeded into all wells containing effector cells at 1 ten thousand per well, and control wells whose aim was to measure only background death. The plates were then briefly centrifuged and incubated at 37 ℃ and 5% CO₂Incubate for 4 hours. The plates were then centrifuged, the supernatant aspirated, and the cells resuspended in PBS containing propidium iodide to measure cell death. The cells were then run through an intellicytic iQue screener plus and the proportion of target cells (separated from the effector region by their staining) that were also positive for PI staining was measured. The percentage of dead cells was then compared to the number of naturally dead cells in control wells and the percentage of cells specifically killed by effector cells was calculated. The results are shown in FIGS. 4 and 5. FIG. 4 shows the equivalent cytotoxicity of CCR7 upregulated clone against K562 cells compared to the parental cell line, and FIG. 5 shows the equivalent cytotoxicity of CCR7 upregulated clone against HL-60 cells compared to the parental cell line.

In vitro testing consists of using a Boyden chamber assay and Matrigel layer to prevent migration. In these assays, modified cells expressing CCR7 showed migration to CCL21 and CCL19, an alternative CCR7 ligand. Cells were placed in the upper well and a thin layer of Matrigel (ECM-like matrix) coated on 8uM pores was separated from the lower chamber. Cells (2.5 ten thousand per well) were placed in low serum medium (supplemented with 1% human serum and 500U/mL IL-2) in the upper chamber and used in the lower chamber alone or containing the chemokine of interestThe same low serum medium. In this case, CCL21 was used at 15ng/mL, CCL19 was used at 15ng/mL, and SDF-1a was used at 20 ng/mL. For the

Modified cells or cells expressing CCR7

Cells, each test was performed in triplicate. The plate was then placed in an incubator overnight for 18 hours of invasive assay, after which the upper chamber was removed and 150 μ L (total volume 750 μ L) was sampled from the lower well after thorough mixing and read on a MacsQuant FACS analyzer. Viable cells in the lower chamber were counted and the cell number was then compared to wells without chemokines and an invasiveness index number was generated. These numbers were averaged and statistical correlations were calculated using the two-tailed t-test. Since the lower wells were sampled without cells detaching from the lower membrane, those cells still attached to the lower part of the ECM would not be represented in these numbers, possibly creating a difference between CCL19 and CCL 21. The results are shown in FIG. 7. In particular, FIG. 7 shows modified expression of CCR7

Statistical significance was increased in the invasiveness of cells to CCL19, a CCR7 chemokine. The lack of a statistically significant response to CCL21 may be due to the nature of the assays performed. This assay measures invasion and subsequent detachment from the ECM, a behavior consistent with the gradient migration of CCL 19. CCL21 does not induce detachment from the matrix while inducing migration, requiring additional steps to demonstrate statistically significant invasive potential.

Example 2. production of NFAT responsive constructs for controlled expression of CCL 21.

To identify NFAT responsive elements, the linearized construct was electroporated into

To produce stable earth surface in cellsCell lines expressing the NFAT-based luciferase expression cassette (NR2.2), these

The cell contains a termination region, followed by 3 NFAT responsive elements (SEQ ID NO: 4), and a minimal promoter (SEQ ID NO: 5), so that the minimal promoter will drive the production of firefly luciferase in the presence of activated NFAT. Then, the cells were also used with a composition containing anti-CD 19CAR (an antigen present on Sup-B15 cells, otherwise to

Cell killing resistant) to electroporate a subset of these cells. These cells are indicated in the left panel as ENR 2.2. Cells were then seeded in triplicate in the absence or presence of target cells and incubated for 2.5 hours to 24 hours. At the end of the incubation period, step 1 reagents from the Promega DualGlo system were added to the wells to activate luciferase (activated by providing its substrate luciferin). The results were then read on a SpectraMax i3x microplate reader and expressed as the mean of the standard deviations calculated in Microsoft Excel. The results are shown in FIGS. 6A and 6B. NFAT activation was demonstrated where the target bound to K562 in a time-dependent manner and only bound to Sup-B15 when electroporated with mRNA of CD 19-CAR.

Example 3. modified NK cell lines expressing CCR7 and CCL 21.

Incorporation of pCRENFAT-CCL21 plasmid into CCR 7-containing construct using LoxP sites embedded in pNKAT-CCR7-LP3 construct in recombinase-mediated cassette exchange

In the cell. Following electroporation of the circular plasmid (pCRENFAT-CCL21), Cre recombinase is transiently expressed, mediating the exchange of the new LoxP flanking cassette with the old selection cassette. The incorporation of a new cassette was facilitated using selection in blasticidin and monoclonal cell lines were subcloned from the resulting population in the same manner as previously described in example 1 to obtain modified CCR7 and CCL21 expressing

A cell.

To evaluate modified expressing CCR7 and CCL21

Cells, modified unstained

Cells were co-cultured with cells known to cause NFAT activation (K562 or other cell lines) in the lower well of the Boyden chamber and stained modified

The cells are placed in the upper chamber. The system is functioning if it is demonstrated that migration is induced by co-culture with a sensitive cell line.

Example 4: in vitro cytotoxicity assays using modified NK cell lines expressing CCR7

FIG. 9 shows modified to express CCR7, as described in example 1

The cells remained cytotoxic to the target cells after migration in a modified Boyden chamber transwell assay.

Example 5: CCR7, CD16, and CD19CAR in transfection with nucleic acid constructs

Cell surface expression in cells

FIG. 12 shows modified antibodies transfected with nucleic acid constructs encoding CCR7, CD16, and CD19CAR

The cells express high levels of the corresponding protein on the cell surface.

Example 6: biodistribution of chemokine-responsive NK cells expressing CCR7 in NSG mice harboring CCL19 positive subcutaneous K562 tumors

This study demonstrated that chemokine-responsive aNK cells home to chemokine-expressing target tissues following intravenous administration. Because mouse and human chemokines do not cross-react, the inventors developed tumor models expressing local ligands as surrogate models.

Experimental method (table 1):

a. animals:

i. animal type: NSG mice (JAX), female, 7-8 weeks old

Animal number: 30 (28 animals received NK cell injection [24 +4 additional ]; another 2 mice received no NK treatment and used as a flow cytometry negative control)

b. Tumor model:

i. cell line: k562, parental and subline expressing CCL19, K-19

A route of vaccination: subcutaneous injection; the parent K562 was inoculated in the left flank, K-19 in the right flank

inoculum: 1E6 cells in 100. mu.L serum-free Medium/Matrigel (v/v 1: 1)

Tumor burden at start of treatment: k-19 average 107mm³(ii) a Parent K562 average 135mm³

v. random grouping:Primaryanimals were randomly grouped according to the volume of K-19 tumors.

c. And (3) testing the sample:

nk cells:

CD19t-haNK (nonc) (Nantkwest Torrey Pines, Western south China)

2. Tetracistronic Mi-aNK R7-19.1 (NantKwest Woburn)

Fluorescent labeling:

1. both types of NK cells were labeled with CFSE according to the manufacturer's manual, immediately prior to in vivo administration.

2. For each time point, cultured CFSE labeled cells were harvested for use as a positive control for flow cytometry.

Method of application: intravenous administration of drugs

Dosage:

1.1E 7 cells/mouse

v. dosing frequency: single dose

d. Tumor collection:

i. time points are as follows: 3, 24 and 48 hours (. + -. 2 hours) after administration

ii.n ═ 4 mice/group/time point

Tumor treatment: the collected tumors were dissociated into single cell suspensions according to the internal protocol (additional) and flow cytometric counting of CFSE positive NK cells was performed.

e. Formulas and statistical analysis:

i. tumor volume is length x width²2 (Length and Width are the longest and shortest diameters of tumors, respectively)

Statistical analysis was performed by one-way analysis of variance followed by multiple comparisons by Tukey's test using GraphPad Prism version 7.0. P < 0.05 was considered statistically significant.

TABLE 1 Experimental setup

As a result:

a. safety:

animals receiving both types of NK cells showed mild to moderate acute reactions immediately after cell infusion (G1-G2, mild-marked depression, lethargy, slow or no response).

Within 24 hours after injection, 2 animals (14 total) of the R7-19.1 group were found dead, while none of the CD19t-haNK group was found dead.

After v.24h, animals of the CD19t-haNK group recovered, while animals of the R7-19.1 group continued to exhibit mild depression and a weak response to stimulation (G1). They became more responsive at 48 hours (G0), but still appeared coarse in coat and breathy.

NK cell homing:

the number of tumor infiltrating NK cells per time point is listed in table 2 and is plotted in figure 13.

1. The combination of the four CCR7 receptor-CCL 19 ligands is:

a.CD19

k562 tumor: receptor-free ligand-free-]；

b.CD19

K-19 tumors: no receptor-with ligand [ - +]；

R7-19.1 cells, K562 tumor: with receptor-without ligand [ + - ]; and

r7-19.1 cells, K-19 tumors: having receptors-having ligands [ + ]

At 3 hours, the homing of R7-19.1 cells to K-19 tumors [ + + ] was significantly greater than that of non-CR CD19t-haNK cells to CCL 19-or CCL19+ tumors [ - - ] and [ + ], respectively; P < 0.05). However, since we could not recover many cells from 2 of the four K562 tumors of group B, a direct comparison of homing of R7-19.1 cells to CCL19 negative and positive tumors in the same animal could not be achieved.

At 24 hours, there was no statistically significant difference in NK cell homing for any of the four CCR7 receptor-CCL 19 ligand combinations. However, when comparing homing of each NK cell line to CCL19+ and-tumors in the same animal, 3 of 4 animals receiving R7-19.1 cells showed improved infiltration into CCL19+ tumors, while most received CD19

The animals showed similar levels of NK infiltration into both tumors regardless of their CCL19 expression (figure 14).

At 48 hours, the total number of tumor infiltrating NK cells decreased in all combinations and there was no difference between any groups.

Table 2. homing of non-CR and R7-19.1 to parental tumors or CCL19 expressing tumors at the indicated time points. Results are mean ± SEM. Unless otherwise stated, N-4.

*，N＝2。

This example demonstrates that the coexistence of CCR7 receptor and CCL19 ligand results in more efficient NK cell infiltration 3 hours after administration. NK cells showed similar levels of tumor homing, regardless of CCL19 expression status, in 3 of 4 animals, in animals receiving non-CR aNK cells 24 hours after dosing. In contrast, NK cells were able to home to CCL19 positive tumors more efficiently in 3 of 4 animals, among animals receiving R7-19.1 cells, compared to the parental control that did not express the ligand. Regardless of receptor or ligand expression, the number of tumor infiltrating NK cells decreased at 48 hours. Especially in the early hours (< 48 hours post-dose) there was a tendency for significantly more homing of R7-19.1 cells to CCL19 tumors, indicating that higher exposure and possibly also stronger cytotoxicity if the cells were used in a therapeutic setting.

Example 7: comparative efficacy evaluation of R7-19.1 cells expressing CCR7 in NSG mice bearing intravenous CCL19 positive RAJI tumors

CCR7 is a chemokine receptor that induces migration of cells towards a gradient of the chemokines CCL19 and CCL21, which are commonly expressed in lymph nodes and other lymphoid organs that are the major sites for B-cell lymphoma disease manifestations. We have created chemokine responsiveness to express CD19-CAR, CCR7, CD16.158V and ERIL-2 based on the t-hanK platform

Cells (i.e., R7-19.1 cells). In addition to CCR7 expression, these cells also have cancer-targeting Chimeric Antigen Receptors (CARs) against CD19 cancer antigen, CD16 variants, and ER-IL-2. Previous profiling studies have demonstrated that R7-19.1 cells preferentially home to Subcutaneous (SC) tumors expressing CCL19 compared to the parental counterpart.

In this study, the anti-tumor effect of repeated intravenous administration of R7-19.1 cells was evaluated in NSG mice in an Intravenous (IV) xenograft model of Raji-19.5, Raji-19.5 being a Raji human Burkitt lymphoma cell engineered to express CCL 19. CD19t-haNK cells that do not express CCR 7(

[ anti-CD 19-CAR, CD16.158V, ERIL-2]) Used as a control NK cell line. Vehicle controls were also included.

Although both NK cell lines demonstrated significant therapeutic efficacy in prolonging survival of IV Raji-19.5 tumor-bearing animals compared to vehicle controls, treatment with R7-19.1 cells gave significantly higher survival benefits than CD19t-haNK cells. Although clear treatment-related responses were observed in both NK cell lines, it is speculated that these responses are a mouse-specific problem associated with the use of relatively high doses of human-derived cells.

Basic principles and purposes of research: in vivo distribution data showed that IV administered R7-19.1 cells showed increased homing to SC tumors expressing CCL 19. In this study, the anti-tumor effect of repeated IV administration of R7-19.1 cells was evaluated in NSG mice in an IV xenograft model of Raji-19.5 (a subline of Raji expressing CCL 19). Note that the original study protocol contained other animal groups not included in the report (groups a-C). They were not relevant for efficacy determination of R7-19.1 cells in this tumor model (for a simplified experimental design, see Table 3).

Study materials:

one or more test articles. The test samples were R7-19.1 cells and CD19t-haNK cells (control NK cells not expressing CCR 7; clone 6). R7-19.1 cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% Pluronic F68. For CD19t-haNK cells (control NK cells that do not express CCR 7; clone 6), CD19t-haNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% pluronic F68. Serum-free growth medium was used as a vehicle control.

The test system comprises: test animals: Cg-Prkdc using NOD.10-11 weeks of age at study start (after quarantine and acclimation) and a body weight between 20-27 grams at randomized cohort^scidIl2rg^tmIWjlSzJ (NSG) female mice. The number of animals used in The study was 30 and The supplier was The Jackson Laboratory (The Jackson Laboratory) (balbour Street 610, maine, usa, zip code 04609(610 Main Street Bar Harbor, ME 04609 US)). Animals were identified with ear tags, cage numbers and tail numbers.

Raji-19.5 tumor model (cancer cell line)

Cell culture medium: raji-19.5 cancer cells were grown in modified RPMI-1640 medium prepared with ATCC supplemented with 10% fetal bovine serum.

Cell harvesting: according to SOP _ Suspension Cancer Cell Collection for In Vivo Studies of southwest Western Inc. [ Collection of suspended Cancer cells for In Vivo Studies]Exponential phase Raji-19.5 cells (passage 16) were collected by centrifugation. The cells were then washed and washed at 5X 10⁵The concentration of individual cells/mL was resuspended in serum-free medium and stored on ice prior to animal inoculation. The cells used in the in vivo study had 97% viability.

Inoculation: 30 mice were inoculated with a volume of 0.2mL of 1X 10 via the lateral tail vein⁵And (4) cells. This is defined as day 0.

Experimental procedures

Weight: animals were weighed prior to enrollment (after quarantine/acclimation), prior to randomization, on the day of each dose (but before dosing), the next day after each dose and prior to euthanasia. Animals exhibiting > 20% weight loss compared to baseline (day 0) weight were euthanized according to IACUC institutional policy, and necropsy was then conducted at the discretion of the investigator.

And (3) clinical observation: animals were observed daily for mortality/morbidity (G0-G4; see table 2 in the study protocol of appendix 1). Moribund and paralyzed animals were euthanized and necropsied as determined by the investigator.

And (3) random grouping: on day 3 (3 days after tumor cell inoculation), 30 tumor-bearing mice were pseudo-randomly assigned to 3 study groups of 10 mice each, based on animal body weight.

Application of the test sample: r7-19.1 and control CD19t-haNK cells growing in exponential phase were harvested by centrifugation twice weekly for 4 consecutive weeks (on

days

3, 6, 10, 13, 17, 20, 24 and 27) and cultured at 5X 10⁷The concentration of individual cells/mL was formulated in serum-free growth medium to pass through an injection volume of 200. mu.L at 1X 10 per mouse⁷Individual cell doses were administered IV. All cell handling and formulation procedures were performed at room temperature. Cell viability was higher than 80% for all dose formulations. As shown in Table 3, group E and group F received CD19t-hanK and R7-19.1 cells, respectively, concurrently with group D receiving the vehicle control.

End point: animals are euthanized when paralyzed, moribund, or meet any other endpoint criteria defined by the agency IACUC. The experiment was terminated on day 30 when the last surviving animal died from the disease. Euthanasia is via inhalation of CO₂And then by cervical dislocation. Some euthanized animals were necropsied to identify tumor nodules visible on internal organs, within the discretion of the study director. A complete record of mortality and death events and autopsy results can be found in appendix 5.

Table 3: research design (simple)

BIW: twice a week; IACUC: institutional animal care and use committee; IV: intravenously.

And (3) data analysis:

body weight curve: the weight curves were analyzed by two-way analysis of variance (or by mixed effects in the presence of missing values; see revision 2 of appendix 1) followed by multiple comparisons by Tukey's test.

Survival curves: survival curves were analyzed by log rank (Mantel-Cox) test.

Statistical analysis: all statistical analyses were performed using GraphPad Prism version 8. P < 0.05 was considered statistically significant.

Results

The efficacy is as follows: the primary indication of efficacy is animal survival. When animals were euthanized by morbidity, paralysis, or weight loss > 20%, mortality events were counted. No animal death was found. As shown in figure 15 and table 4, treatment with both R7-19.1 and control CD19t-haNK cells significantly prolonged survival of Raji-19.5 IV tumor-bearing animals compared to vehicle control (P < 0.0001 for R7-19.1 and P ═ 0.0002 for CD19t-haNK by log rank test), with a median survival increase of 6.5 days and 2.5 days, respectively. This corresponds to a 30% and 12% increase relative to vehicle control, respectively. More importantly, expression of the CCR7 chemokine-responsive receptor in R7-19.1 cells increased median survival by an additional 4 days (17%) compared to CD19t-haNK cells (P < 0.0001).

Table 4: median survival summary of Raji-19.5 IV tumor-bearing NSG mice treated with vehicle, CD19t-hanK cells or R7-19.1 cells.

NA: not applicable.

Safety feature

As shown in figure 16, NK cell treated animals consistently exhibited 5% -10% weight loss after each treatment administration. In most cases, animals were able to recover from cell injections and showed weight recovery, causing oscillations in the weight change curve between doses. However, the initial dose appears to elicit the most severe response and is associated with the clinical symptoms and the longest recovery time for weight changes. Such responses are not uncommon in animals receiving IV NK infusions and certainly are not R7-19.1 cell specific. Recovery of body weight indicates that weight loss is temporary and reversible.

Notably, at the end of the study, the animals showed a dramatic drop in body weight (fig. 16), which may be due to disease progression. Necropsy revealed tumor nodules in the liver, ovary and occasionally spleen of almost all animals examined. One exception was the first mouse euthanized in the group of CD19t-haNK cells, which reached > 20% weight loss the day after the 6 th dose. This animal did not identify visible tumor nodules during necropsy. Therefore, the exact cause of weight loss that warrants euthanasia cannot be determined.

Conclusion

In the IV Raji-19.5 xenograft model, at 1X 10⁷Dosing level of individual cells/dose IV administration of freshly prepared R7-19.1 cells (twice weekly for 4 weeks) showed significant and statistically significant antitumor efficacy. This treatment provided a median survival increase of 6.5 days or 30% relative to the vehicle control group and a 4 day or 17% increase relative to the CD19t-haNK treated group.

Although clear treatment-related responses were observed, these responses were transient and surviving animals did show signs of recovery. These responses are presumed to be a mouse-specific problem associated with administration of relatively high doses of human NK cells and therefore unlikely to be transformed into humans.

Overall, R7-19.1 cells expressing CCR7 showed significant therapeutic efficacy in this IV model of Raji-19.5 compared to vehicle and its non-chemokine responsive counterpart.

Example 8: comparative efficacy evaluation of R7-19.1 cells expressing CCR7 in NSG mice bearing subcutaneous CCL19 positive RAJI tumors

CCR7 is a chemokine receptor that induces migration of cells towards a gradient of the chemokines CCL19 and CCL21, which are commonly expressed in lymph nodes and other lymphoid organs that are the major sites for B-cell lymphoma disease manifestations. We have created chemokine responsiveness to express aCD19-CAR, CCR7, CD16.158V and ERIL-2 based on the t-hanK platform

In this study, the anti-tumor effect of repeated Intravenous (IV) administration of R7-19.1 cells was evaluated in NSG mice in an SC xenograft model of Raji-19.5, Raji-19.5 being a Raji human Burkitt lymphoma cell engineered to express CCL 19. CD19t-haNK cells that do not express CCR 7(

In a subset of tumor-bearing animals, both NK cell treatments showed significant effects in inhibiting tumor growth, with R7-19.1 treatment exhibiting greater inhibition than control CD19t-haNK cells. Although clear treatment-related responses were observed in both NK cell lines, it is speculated that these responses are a mouse-specific problem associated with the use of relatively high doses of human-derived cells.

Study of the basic principles and purposes: in vivo distribution data showed that IV administered R7-19.1 cells showed increased homing to SC tumors expressing CCL 19. In this study, the anti-tumor effect of repeated IV administration of R7-19.1 cells was evaluated in NSG mice in IV and SC xenograft models of Raji-19.5 (a subline of Raji expressing CCL 19).

Research materials

One or more test items: r7-19.1 cells (clone: and CD19t-haNK cells (control NK cells that do not express CCR 7; clone 6) were used as test samples, while growth medium was used as vehicle control.

R7-19.1 cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% Pluronic F68.

CD19t-haNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% pluronic F68.

The test system comprises: 30 NOD.Cg-Prkdc animals 10-11 weeks old at study start (after quarantine and acclimation) and 19-28 grams in weight on tumor transplantation day were used in the study^scidIl2rg^tmIWjlSzJ (NSG) female mice. The supplier was Jackson laboratory (Balhong street 610, Mine, USA, zip code 04609). Animals were identified using ear tags, cage numbers and tail numbers.

Raji-19.5 tumor model (cancer cell line)

Cell culture medium: raji-19.5 cancer cells were cultured in modified RPMI-1640 medium prepared with ATCC supplemented with 10% fetal bovine serum.

Cell harvesting: exponential phase Raji-19.5 cells (passage 16) were collected by centrifugation according to SOP _ of southwest company for collection of suspended cancer cells for in vivo studies. The cells were then washed and resuspended in serum-free medium before mixing with an aliquot of Matrigel to reach 2.5 × 10⁶Final concentration of individual cells/mL. Cells were stored on ice prior to animal inoculation. The cells used in the in vivo study had 97% viability.

Inoculation: 30 animals were unilaterally inoculated with a 100. mu.L volume of 2.5X 10 in the right flank⁵And (4) individual cancer cells. The skin was shaved prior to injection.

Experimental procedures

Tumor volume measurement: after SC implantation into the tumor, the animals were examined at least twice a week for tumor formation. Tumor Volume (TV) was measured twice weekly with a digital hand-held caliper when the tumor became measurable and calculated using the following formula: length x width of TV²/2[ Length is the maximum diameter of tumor, Width is the minimum diameter of tumor]. According to the policy of an organization IACUC, the tumor volume exceeds 2000mm³Or animals with ulcerated tumors were euthanized and necropsied. Tumor Growth Inhibition (TGI) was calculated as follows: TGI ═ T_C-T_t)/ΔT_CX 100% where T_CAnd T_tAre respectively provided withIs the mean tumor volume at a particular time point for the control and treated groups; and Δ T_CIs the change in mean tumor volume of the control group.

Weight: animals were weighed prior to enrollment (after quarantine/acclimation), prior to randomization, on the day of each dose (but before dosing), the next day after each dose and prior to euthanasia. Animals exhibiting > 20% weight loss compared to baseline (day 1) weight were euthanized according to IACUC institutional policy, followed by necropsy.

And (3) clinical observation: animals were observed daily for mortality/morbidity (G0-G4; see Table 5). Moribund or paralyzed animals were euthanized and necropsied.

And (3) random grouping: when the average tumor volume reaches 190mm³At the time, 30 mice were pseudo-randomly divided into 3 study groups of 10 mice each to achieve similar tumor volumes between groups. This is defined as day 1. Notably, each group contained two subpopulations due to the large variability in tumor size when randomly grouped: 4-5 animals had large (> 200 mm)³) Tumors, 5-6 with small (< 200 mm)³) Tumor (see fig. 17).

Application of the test sample: r7-19.1 and control CD19t-haNK cells grown in exponential phase were harvested by centrifugation twice weekly for 4 consecutive weeks (on

days

1, 4, 9, 12 and 15) and at 5X 10⁷The concentration of individual cells/mL was formulated in serum-free growth medium to pass through an injection volume of 200. mu.L at 1X 10 per mouse⁷Individual cell doses were administered IV. All cell handling and formulation procedures were performed at room temperature. Cell viability was higher than 80% for all dose formulations. As shown in Table 5, group A received vehicle control, while group B and group C received CD19t-hanK and R7-19.1 cells, respectively.

End point: when the animal reaches any of the above endpoints defined by the agency IACUC, the animal is euthanized. Euthanasia is via inhalation of CO₂And then by cervical dislocation. Some euthanized animals were necropsied to identify tumor nodules visible on internal organs, as determined by the study director.

Table 5: research design (simple)

BIW: twice a week; IACUC: institutional animal care and use committee; IV: intravenously; TGI: tumor growth inhibition.

¹IACUC defined tumor burden endpoint: tumor volume over 2000mm³(ii) a Ulcerating tumors; and tumors interfering with normal gait.

Data analysis

Calculating the tumor volume: tumor volume is length x width²2 (Length and Width are the longest and shortest diameters of tumors, respectively)

Tumor Growth Inhibition (TGI) calculation: TGI ═ T_C-T_t)/ΔT_CX 100% where T_CAnd T_tMean tumor volumes at specific practical points for control and treatment groups, respectively, and Δ T_CIs the change in mean tumor volume in the control group.

Statistical analysis of tumor growth and body weight curves: tumor growth and weight curves were analyzed by two-way analysis of variance (or by mixed effects in the presence of missing values; see revision 2 of appendix 1) followed by multiple comparisons by Tukey's test. All statistical analyses were performed using GraphPad Prism version 8. P < 0.05 was considered statistically significant.

Results

The efficacy is as follows: the primary readout in this study was tumor growth inhibition. SC tumors with different initial tumor volumes may have different developed vasculature and CCL19 gradients, which may affect the distribution of the test article to the tumor. In addition, small and large tumors can respond to NK cell processing in different ways. For these reasons, two subpopulations (i.e. large and small tumors) were analyzed separately.

As shown in FIG. 18, two animals were used in a subpopulation of animals with large initial tumors when compared to vehicle controlThe seed NK cell line treatments all showed significant tumor growth inhibition due to tumor volumes exceeding 2000mm on day 9³Or the presence of ulcerated tumors, 3 out of 4 animals had to be euthanized. In contrast, in the NK cell treatment group, most animals survived to day 12 or day 15. More importantly, at day 15, tumor growth inhibition was clearly enhanced in groups R7-19.1, with a TGI of 35%, compared to the counterpart not expressing CCR 7. This difference failed to reach statistical significance, probably due to the smaller size of the cohort (N for CD19t-haNK and R7-19.1 groups were 2 and 4, respectively).

However, for animals with small tumors at the start of treatment, neither NK cell therapy was effective at inhibiting tumor growth compared to vehicle control, or to each other (fig. 19). This may be due to three factors: 1) the vascular system of small tumors may not be fully developed and thus have less exposure/accessibility to the test article, resulting in the ineffectiveness of both NK cell therapies. 2) CCR 7-mediated chemotaxis requires a chemokine gradient, rather than responding only to the presence of chemokines. Such a CCL19 gradient may be insufficiently established in small tumors, resulting in a lack of distinguishability between R7-19.1 cells and CD19t-haNK controls. And 3) in these already small cohorts, there were some "outliers" in tumor growth, further excluding the possibility of statistical analysis.

As shown in figure 20, NK cell treated animals typically exhibited 5% -10% weight loss after each treatment administration. In most cases, animals were able to recover from cell injections and showed weight recovery, causing oscillations in the weight change curve between doses. However, the initial dose appears to elicit the most acute and severe response and is associated with the clinical symptoms and the longest recovery time for weight changes. Such responses are not uncommon in animals receiving IV NK infusions and certainly are not R7-19.1 cell specific. Recovery of body weight indicates that weight loss is temporary and reversible.

Conclusion

At 1 × 10⁷Individual cell/agent administration level pairIV administration of freshly prepared chemokine-responsive R7-19.1 cells twice weekly for 4 weeks resulted in significant inhibition of tumor growth in animals bearing large SC Raji-19.5 tumors, but such effects did not reach statistical significance, probably due to the smaller size of the cohort. This treatment regimen showed no therapeutic efficacy in animals with smaller initial tumor volumes. This may be due to inadequate development of tumor vasculature and/or inadequate establishment of chemokine gradients in small tumors. A clear treatment-related reaction was observed. However, these reactions were transient and the animals did show signs of recovery. These responses are presumed to be a mouse-specific problem associated with administration of relatively high doses of human NK cells and therefore unlikely to be transformed into humans.

Example 9: production of aTGFβ/PD-L1 CAR-modified

Cells

The TGF β trap, consisting of a single-chain dimer of the extracellular domain of TGF β RII, was cloned into a tetra-cistronic plasmid vector that also contained the PD-L1 CAR, CD16, and erll-2 transgenes (figure 21). The tetra-cistronic plasmid was electroporated into aNK cells to generate modified NK-92 cells. Selection of modified by IL-2 deficient media

Cells, because IL-2 dependent untransformed aNK cells cannot survive in IL-2 deficient medium. FIG. 22 shows aTGFβ/PD-L1 CAR-modified

The cells co-expressed high levels of PD-L1 CAR and CD16.

Restriction dilution cloning

Polyclonal aTGF beta/PD-L1 t-haNK^TMAliquots of the library cultures were diluted to a density of 3 cells/ml in growth medium without IL-2 addition. The cell suspension was aliquoted into 96-well plates at a volume of 200. mu.l per well, which corresponds to a flat per wellAll 0.6 cells. The plates were incubated at 37 ℃ for 10 days and then visually inspected for cell growth. Clones were picked and transferred to larger containers for further amplification and characterization.

Biological analysis method

Cell culture:

cloning and cloning of aTGF beta/PD-L1 t-haNK^TMCells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and free of IL-2.

aNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 500IU/ml recombinant human IL-2.

haNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and free of IL-2.

K562 and MDA-MB-231 cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum and an antibiotic/antifungal cocktail. K562 cells were passaged every 2-5 days or every time the medium appeared yellow.

Will SUP-B15^PD-L1+And SUP-B15^CD19KO/CD20+Cells were cultured in RPMI-1640 supplemented with 20% heat-inactivated fetal bovine serum, 55uM β -mercaptoethanol and an antibiotic/antifungal mixture. The cells were passaged as for the K562 cells described above.

Antibody staining for flow cytometry analysis:

cells were harvested by centrifugation, washed twice in FACS buffer (5% FBS in 1X D-PBS) and resuspended in 1ml FACS buffer. For direct fluorophore-conjugated antibody staining of surface proteins, cells were incubated with the appropriate conjugated antibody (or isotype control) at 4 ℃ for 20 min in the dark, then washed twice with FACS buffer. To detect CAR proteins, cells were incubated with biotinylated anti-F (ab')₂Fragment antibodies were incubated with subsequent streptavidin-APC antibodies. Samples were analyzed on a macSQurant flow cytometer.

Cytotoxicity:

cell lines grown in suspension were resuspended by pipetting the cell culture up and down. Cell viability was determined by automatic counting (trypan blue exclusion). Target cells were labeled with CFSE dye and diluted to the desired cell concentration with effector cells in RPMI-1640 supplemented with 10% heat-inactivated FBS and antibiotic/antifungal. The effector and target cells were mixed in 96-well plates at different effector to target (E: T20: 1, 10: 1, 5: 1, 2.5: 1, 1.25: 1, 0.62: 1, 0.31: 1 and 0.15: 1) ratios and incubated for 4h in an incubator at 37 ℃ under 5% CO2 atmosphere. PI was then added to fluorescently label dead cells and the assay was analyzed on a MACSquant flow cytometry apparatus.

ADCC：

Cell lines grown in suspension were resuspended by pipetting the cell culture up and down. Cell viability was determined by automatic counting (trypan blue exclusion). Target cells were labeled with PKH67-GL dye and diluted to the desired cell concentration with target and effector cells in RPMI-1640 supplemented with 10% heat-inactivated FBS and antibiotic/antifungal agent. The target cells were preincubated with the monoclonal antibodies trastuzumab, rituximab or no antibody for 30min at room temperature. Antibody-labeled target cells (and no antibody controls) and effector cells were then mixed in 96-well plates at different effector to target ratios (E: T of 20: 1, 10: 1, 5: 1, 2.5: 1, 1.25: 1, 0.62: 1, 0.31: 1, and 0.15: 1) and at 5% CO₂Co-incubation was carried out for 4h at 37 ℃ in an incubator under atmosphere. PI was then added to fluorescently label dead cells and the assay was analyzed on a MACSquant flow cytometry apparatus.

Quantification of TGF-beta trap secreted by aTGF-beta/PD-L1 t-haNK cells

The sample supernatant for analysis was prepared by removing cells by a first centrifugation step at 500x g for 5min, followed by removal of cell debris by a second centrifugation step at 2000Xg for 5 min. The sample supernatant was frozen at-80 ℃ until analysis. Cell pellets from 500xg centrifugation steps were resuspended, pooled in triplicate and cell density recorded. User of the inventionTGFβRIIELISA detection kitMeasuring in the supernatant of the sample according to the manufacturer's instructionsTGF beta trapConcentration, and comparison to the standard provided. Will be provided withTGF beta trapConcentrations were normalized to cell number and expressed as pg/ml/10⁶And (4) cells. FIG. 23 shows that all a ⁶TGF beta PD-L1t-haNK clones all secreted large amounts of TGF beta trap (between 6 and 13ng/ml/10 thin lines) Between cells).

^TMCytotoxicity of aTGF beta/PD-L1 t-haNK cells on target cell lines

aTGFβ/PD-L1 t-haNK^TMCytotoxicity of cells was determined by contacting the cells with the target cells K562 cells, SUP-B15^PD-L1+Cells were incubated with MDA-MB-231 cells for analysis. FIG. 24 shows aTGF β/PD-L1t-hanK upon killing K562 cells (target cells)^TMThe cells maintained cytotoxicity equivalent to that of the parent aNK cells.

FIG. 25 shows aTGF β/PD-L1t-haNK^TMCell display vs. aNK^TMSpecific killing enhancement of the resistant, PD-L1 positive SUP-B15 cell line-at an effector to target ratio of 8, about 70% of the cells are killed by aTGF β/PD-L1t-haNK relative to only about 10% of the cells being killed by aNK cells^TMAnd (4) killing the cells.

FIG. 26 shows aTGF β/PD-L1t-haNK^TMCells exhibit enhanced specific killing of the MDA-MB-231 cell line-at an effector to target ratio of 8, about 90% of the cells are killed by the aTGF β/PD-L1t-haNK cells relative to only about 40% of the cells being killed by the aNK cells^TMAnd (4) killing the cells.

FIG. 27 shows aTGF β/PD-L1t-haNK^TMCombination of cells with anti-CD 20 rituximab monoclonal antibody or anti-Her 2-neu trastuzumab monoclonal antibody against SUP-B15^CD19KO/CD20+ADCC Activity of cells (CD19-, CD20+, Her2-neu-, NK-resistant). aTGF beta/PD-L1 t-haNK cells were able to effectively target and kill resistant SUP-B15 when combined with the anti-CD 20 antibody rituximab in a 4h cytotoxicity assay^CD19KO/CD20+. When combined with the anti-Her 2/neu control antibody trastuzumab,

and aTGF beta/PD-L1 t-haNK^TMNone of the clones killed the target SUP-B15^CD19KO/CD20+A cell.

Example 10:modified

Secreted by cellsTGFβWell suppressionTGF-beta Activity

Engineered HEK293 cells directly expressing the TGF β responsive element (SMAD binding promoter) of the luciferase reporter showed a dose-dependent increase in luciferase activity when treated with TGF β (fig. 28).

Figure 29 shows that induction of luciferase activity by TGF β in HEK293 reporter cells can be inhibited by co-incubation with culture supernatant of aTGF β/PD-L1t-haNK cells, whereas culture supernatant from haNK control cells has limited effect on luciferase activity.

Example 11: modified

Cellular production of IL-12

NK-92 cells were transduced with lentiviral constructs in the p35-p40 orientation or the p40-p35 orientation encoding a functional IL-12 p70 dimer in the form of a single chain polypeptide (FIG. 30). Following neomycin selection, transduced NK-92 cells were able to secrete detectable levels of p70 IL-12. The addition of 2A peptide at the C-terminus of the IL-12 dimer did not affect protein secretion.

IL-12/PD-L1 CAR-modified

Production of cells

The single chain dimer of IL-12 (scIL-12 p70) was cloned into a tetra-cistronic plasmid vector that also contained the PD-L1 CAR, CD16 and erll-2 transgenes. The tetra-cistronic plasmid (fig. 31) was electroporated into aNK cells to generate modified NK-92 cells. Selection of modified by IL-2 deficient media

Cells, because IL-2 dependent untransformed aNK cells cannot survive in IL-2 deficient medium. FIG. 33 shows IL-12/PD-L1 CAR-modified

The cells were able to secrete large amounts of scIL-12 p70 cytokine.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence accession numbers, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. Accordingly, the inventive subject matter is not to be restricted except in light of the attached claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the claims of this specification refer to at least one of something selected from the group consisting of A, B, c.

The claims (modification according to treaty clause 19)

1. A modified NK-92 cell comprising a nucleic acid encoding a homing receptor operably linked to a promoter, and further comprising a nucleic acid encoding an antigen-binding protein operably linked to a promoter, wherein the antigen-binding protein comprises a Chimeric Antigen Receptor (CAR), and wherein the CAR specifically binds CD19, or has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 25, or a variant thereof, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

2. The modified NK-92 cell of claim 1, wherein the homing receptor is a G protein-coupled receptor (GPCR), a chemokine receptor, a cytokine receptor, a cell adhesion molecule, a selectin, or an integrin.

3. The modified NK-92 cell of claim 2, wherein the chemokine receptor is selected from the group consisting of CCR7, CXCR2, or CXCL14 receptor, and the cell adhesion molecule is selected from the group consisting of L-selectin (CD62L), α 4 β 7 integrin, LPAM-1, and LFA-1.

4. The modified NK-92 cell of claim 3, wherein the nucleic acid encoding CCR7 hybridizes with SEQ ID NO: 1 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

5. Cancellation

6. Cancellation

7. Cancellation

8. Cancellation

9. Cancellation

10. The modified NK-92 cell of any one of claims 1 to 3, wherein the modified NK-92 cell further comprises a nucleic acid encoding a cytokine operably linked to a promoter.

11. The modified NK-92 cell of claim 10, wherein the cytokine is IL-2, erll-2, IL-15, erll-15, or a combination thereof.

12. The modified NK-92 cell of claim 10, wherein the cytokine is erll-2 and the nucleic acid encoding erll-2 is identical to SEQ ID NO: 14 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

13. The modified NK-92 cell of claim 10, wherein the cytokine is erll-15 and the nucleic acid encoding erll-15 is identical to SEQ ID NO: 67 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

14. The modified NK-92 cell of any one of claims 1-3 and 10-13, wherein the modified NK-92 cell further comprises a nucleic acid encoding an Fc receptor operably linked to a promoter.

15. The modified NK-92 cell of claim 14, wherein the Fc receptor is CD16 or high affinity CD16(SEQ ID NO: 12) or is a peptide of SEQ ID NO: 13, a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

16. The modified NK-92 cell of any one of claims 1-3 and 10-15, wherein the homing receptor, antigen binding protein, CAR and/or Fc receptor is expressed on the cell surface of the modified NK-92 cell.

17. Cancellation

18. Cancellation

19. Cancellation

20. Cancellation

21. Cancellation

22. Cancellation

23. Cancellation

24. Cancellation

25. Cancellation

26. Cancellation

27. Cancellation

28. Cancellation

29. Cancellation

30. Cancellation

31. Cancellation

32. A method of producing a modified NK92 cell line, the method comprising targeted homologous recombination at a specific locus, wherein the locus is AAVS1, and wherein the AAVS1 locus is homologous to SEQ ID NO: 7 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

33. Cancellation

34. Cancellation

35. Cancellation

36. Cancellation

37. Cancellation

38. Cancellation

39. Cancellation

40. Cancellation

41. Cancellation

42. Cancellation

43. Cancellation

44. Cancellation

45. Cancellation

46. Cancellation

47. Cancellation

48. Cancellation

49. Cancellation

50. Cancellation

51. Cancellation

52. Cancellation

53. Cancellation

54. Cancellation

55. Cancellation

56. Cancellation

57. Cancellation

58. A modified NK-92 cell comprising a nucleic acid encoding a Transforming Growth Factor (TGF) - β trap and a Chimeric Antigen Receptor (CAR) that specifically binds programmed cell death 1 ligand 1(PD-L1) operably linked to a promoter.

59. The modified NK-92 cell of claim 58, wherein the modified NK-92 cell further comprises a nucleic acid encoding a C-X-C chemokine receptor type 4 (CXCR4) or an antigen binding protein operably linked to a promoter, and wherein the antigen binding protein specifically binds a tumor associated antigen.

60. The modified NK-92 cell of claim 59, wherein the tumor-associated antigen is selected from CD19, CD20, NKG2D ligand, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, CSPG-4, IGF1-R, Flt-3, CD276, CD123, BCMA, CD33, B7-H4, or 4-1 BB.

61. The modified NK-92 cell of claim 59, wherein the antigen binding protein is part of a Chimeric Antigen Receptor (CAR).

62. The modified NK-92 cell of claim 58, wherein the modified NK-92 cell further comprises a nucleic acid encoding a cytokine operably linked to a promoter.

63. The modified NK-92 cell of claim 62, wherein the cytokine is IL-2, ERIL-2, IL-15, ERIL-15, IL-12, or a combination thereof.

64. The modified NK-92 cell of claim 62, wherein the cytokine is ERIL-2.

65. The modified NK-92 cell of claim 58, wherein the modified NK-92 cell further comprises a nucleic acid encoding an Fc receptor operably linked to a promoter.

66. The modified NK-92 cell of claim 65, wherein the Fc receptor is a peptide having the amino acid sequence of SEQ ID NO: 12, or a high affinity CD16, or a sequence that is identical to SEQ ID NO: 13, a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

67. The modified NK-92 cell of claim 58, wherein the TGF- β trap comprises a single chain dimer of a TGF- β receptor II extracellular domain.

68. The modified NK-92 cell of claim 58, wherein the CAR that specifically binds to the PD-L1 consists of a sequence identical to SEQ ID NO: 68 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

Statement or declaration (modification according to treaty clause 19)

Statement according to clause 1 of item 19

Claim 1 has been modified to comply with claim 9, and claim 9 has been deemed by the examining authority to be novel, inventive and industrially applicable. Similarly, claim 32 has been modified to comply with claim 34, and claim 34 is believed to be novel, inventive, and industrially applicable by the reviewing authorities. Accordingly, selected dependent claims are revoked or modified to reflect appropriate dependencies.

The independent claims 17-20, 21, 22, 23, 24, 28, 3, 48, 49, 50, 51, 52 and 56 and all dependent claims thereof are eliminated so that there is no practical significance to the refutation of such claims.

The new claims 58-68 specify: the modified NK-92 cells comprise a nucleic acid encoding a Transforming Growth Factor (TGF) -beta trap and a Chimeric Antigen Receptor (CAR) that specifically binds programmed cell death 1 ligand 1(PD-L1) operably linked to a promoter, neither taught nor suggested in the prior art.

Based at least on the foregoing and modifications herein, applicants believe that all pending claims satisfy all the requirements of the PCT regulations.

Conclusion

Claims 1-4, 10-16, 32 and 58-68 are pending in this application. The applicant(s) request(s) that all pending claims be granted.

Claims

1. A modified NK-92 cell comprising a nucleic acid encoding a homing receptor operably linked to a promoter.

5. The modified NK-92 cell of any one of claims 1 to 4, wherein the modified

The cell further comprises a nucleic acid encoding an antigen binding protein operably linked to the promoter.

6. The modified NK-92 cell of claim 5, wherein the antigen binding protein specifically binds to a tumor associated antigen.

7. The modified NK-92 cell of claim 6, wherein the tumor-associated antigen is selected from CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4, or B7-H4.

8. The modified NK-92 cell of claim 5, wherein the antigen binding protein comprises a Chimeric Antigen Receptor (CAR).

9. The modified NK-92 cell of claim 8, wherein the CAR specifically binds CD19, or has an amino acid sequence that is identical to SEQ ID NO: 25, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

10. The modified NK-92 cell of any one of claims 1 to 9, wherein the modified NK-92 cell further comprises a nucleic acid encoding a cytokine operably linked to a promoter.

14. The modified NK-92 cell of any one of claims 1 to 13, wherein the modified

The cell further comprises a nucleic acid encoding an Fc receptor operably linked to the promoter.

16. The modified NK-92 cell of any one of claims 1-15, wherein the homing receptor, antigen binding protein, CAR and/or Fc receptor is at the modified NK-92 cell

Expressed on the cell surface of the cell.

17. A modified NK-92 cell, the modified NK-92 cell comprising one or more nucleic acids encoding: i) a homing receptor, ii) an ABP or CAR that specifically binds to a target antigen, iii) an Fc receptor, and/or iv) a cytokine selected from IL2, IL-15, erll-2, erll-15, or a combination thereof, wherein one or more nucleic acid sequences are operably linked to a promoter.

18. The modified NK-92 cell of claim 17, wherein the homing receptor is a G protein-coupled receptor (GPCR), a chemokine receptor, a cytokine receptor, a cell adhesion molecule, a selectin, or an integrin.

19. The modified NK-92 cell of claim 17, wherein the Fc receptor is CD16 or high affinity CD16(SEQ ID NO: 12) or is a peptide of SEQ ID NO: 13, a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

20. The modified of any one of claims 1-15

A cell wherein the homing receptor, antigen binding protein, CAR and/or Fc receptor is in the modified

Expressed on the cell surface of the cell.

21. A composition comprising the modified NK-92 cell of any one of claims 1-20, and a pharmaceutically acceptable excipient.

22. A kit comprising the NK-92 cell of any one of claims 1-21, and instructions for use.

23. A method of treating a cancer or tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of the modified NK-92 cell of any one of claims 1-20 or the composition of claim 21, wherein administering treats the cancer or reduces the size of the tumor in the subject.

24. A method of reducing cancer metastasis in a subject, comprising administering to the subject a therapeutically effective amount of the modified NK-92 cell of any one of claims 1-20 or the composition of claim 21, thereby reducing cancer metastasis in the subject.

25. The method of claim 23 or 24, wherein the subject is administered every m²Application of 1X 10³To 1X 10¹⁰Individual NK-92 cells.

26. The method of any one of claims 23-25, wherein the NK-92 cells are administered parenterally, intravenously, peritumorally, or by infusion.

27. The method of any one of claims 23-26, further comprising administering to the subject an additional therapeutic agent.

28. An inducible promoter for NK cell selective and targeted secretion, the inducible promoter comprising NFAT transcription factor responsive elements.

29. The inducible promoter of claim 28, wherein the NFAT transcription factor responsive gene is CCL 21.

30. The inducible promoter of claim 28, wherein NFAT is activated by the fcepsilon RI γ pathway through activation of CD19 CAR.

31. The inducible promoter of claim 28, which is operably linked to the promoter of SEQ ID NO: 13 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

32. A method of generating a modified NK92 cell line, the method comprising targeted homologous recombination at a specific locus.

33. The method of claim 32, wherein the locus is AAVS 1.

34. The method of claim 33, wherein the AAVS1 locus hybridizes to SEQ ID NO: 7 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

35. A modified NK-92 cell comprising a nucleic acid encoding a secreted cytokine that modulates a tumor microenvironment, operably linked to a promoter.

36. The modified NK-92 cell of claim 35, wherein the secreted cytokine is IL-12 and/or a TGF- β trap.

37. The modified NK-92 cell of any one of claims 35 to 36, wherein the modified

38. The modified NK-92 cell of any one of claims 35-37, wherein the antigen binding protein specifically binds a tumor associated antigen.

39. The modified NK-92 cell of claim 38, wherein the tumor-associated antigen is selected from CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4, or B7-H4.

40. The modified NK-92 cell of claim 38, wherein the antigen binding protein comprises a Chimeric Antigen Receptor (CAR).

41. The modified NK-92 cell of claim 40, wherein the CAR specifically binds PD-L1, or has an amino acid sequence that is identical to SEQ ID NO: 69 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

42. The modified NK-92 cell of any one of claims 35-41, wherein the modified NK-92 cell further comprises a nucleic acid encoding a second cytokine operably linked to a promoter.

43. The modified NK-92 cell of claim 42, wherein the second cytokine is IL-2, ERIL-2, IL-15, ERIL-15, or a combination thereof.

44. The modified NK-92 cell of claim 42, wherein the second cytokine is erlI-2, or a nucleic acid encoding erlI-2 is substantially identical to SEQ ID NO: 14 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

45. The modified NK-92 cell of any one of claims 35-44, wherein the modified NK-92 cell further comprises a nucleic acid encoding an Fc receptor operably linked to a promoter.

46. The modified NK-92 cell of claim 45, wherein the Fc receptor is CD16 or high affinity CD16(SEQ ID NO: 12) or is a peptide of SEQ ID NO: 13, a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

47. The modified NK-92 cell of any one of claims 35-46, wherein the IL-12 and/or TGF- β trap is secreted and the antigen binding protein, CAR and/or Fc receptor is expressed on the cell surface of the modified NK-92 cell.

48. A modified NK-92 cell, the modified NK-92 cell comprising one or more nucleic acids encoding: i) IL-12 and/or TGF- β trap, ii) ABP or CAR that specifically binds to a target antigen, iii) Fc receptor, and/or iv) a second cytokine selected from the group consisting of erll-2, erll-15, IL-2, IL-15, or a combination thereof, wherein the one or more nucleic acid sequences are operably linked to a promoter.

49. A composition comprising the modified NK-92 cell of any one of claims 35 to 48, and a pharmaceutically acceptable excipient.

50. A kit comprising the NK-92 cell of any one of claims 35 to 49, and instructions for use.

51. A method of treating a cancer or tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of the modified NK-92 cell of any one of claims 35-48 or the composition of claim 49, wherein administering treats the cancer or reduces the size of the tumor in the subject.

52. A method of reducing cancer metastasis in a subject, comprising administering to the subject a therapeutically effective amount of the modified NK-92 cell of any one of claims 35-48 or the composition of claim 49, thereby reducing cancer metastasis in the subject.

53. The method of claim 51 or 52, wherein the subject is administered every m²Application of 1X 10³To 1X 10¹⁰Individual NK-92 cells.

54. The method of any one of claims 51-53, wherein the NK-92 cells are administered parenterally, intravenously, peritumorally, or by infusion.

55. The method of any one of claims 51-54, further comprising administering to the subject an additional therapeutic agent.

56. A method of administering NK cells to an individual, the method comprising administering a first composition of modified NK-92 cells of any one of claims 35-48, and a second composition comprising primary NK cells.

57. The method of claim 56, wherein the first composition comprises modified NK-92 cells comprising nucleic acid encoding IL-12 and/or TGF- β trap operably linked to a promoter.